Spectrophotometric Microdetermination of Copper in Copper Oxidases

Spectrophotometric Determination of Gold, Copper, and Cobalt. M. H. Hashmi , Abdur. Rashid , Mohammad. Umar , and Farooq. Azam. Analytical Chemistry ...
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adopted as the critical level below which an unknown oil is identified as reclaimed, 85% of the known reclaimed oils listed in Table I1 are identified as such. SUMMARY

I n an analytical method for discriminating between virgin and reclaimed automotive engine lubricating oils, a statistical function, Q, of the infrared absorbances, a t 14 wave lengths, of the oils, serves as the discriminant. The method relies on the differences in complexity of composition of most virgin and reclaimed oils for its successfor a number of technical and economic reasons, most reclaimed oils are a com-

plex mixture of crude sources, whereas individual virgin oils, for the most part, are blended from a few crudes. The Q values of the 7 3 virgin oils examined were (with two exceptions) satisfactorily described by a double logarithmic normal distribution, truncated a t the lower end. The point of truncation is estimated a t Q = 294. Below this value an oil is identified as reclaimed, Of the 54 known reclaimed oils examined, 85y0had Q values below the critical value. ACKNOWLEDGMENT

The authors express their appreciation to W. C. McCrone and R. E. Putscher for their many helpful sug-

gestions, and to R. D. Hites, who obtained the infrared spectra. LITERATURE CITED

( I ) Fred, hl., Putscher, R. E., AXAL. CHEM.21, 900 (1949). (2) Oil Gas J . 54, 215 (1956). ( 3 ) Smith, C. A. B., Ann. Eugenics 13, 272-82 (1947). (4) Smith, H. M., Ind. Eng. Chem. 44, 2577 (1952). ( 5 ) Welch, B. L., Biometrika 34, 28-35 (1947).

RECEIVED for review May 31, 1956. .hecepted October 30, 1957. Work sponsored as a joint cooperative venture by t h e Pennsylvania Grade Crude Oil Association with member and nonmember support.

Spectrophotometric Microdetermination of Copper in Copper Oxidases Using Oxalyldihydrazide GEORGE R. STARK and CHARLES R. DAWSON Department o f Chemistry, Columbia University, New York, N. Y.

b A new method is presented for the determination of the copper in copper oxidases using oxalyldihydrazide and acetaldehyde in ammoniacal solution. The method will determine as little as 0.1 y of copper per mi. in the presence of protein with a precision of 2 to 3%. Manganese(l1) interferes somewhat, but cadmium(ll), nickel(ll), magnesium(ll), iron(ll), zinc(ll), cobalt(il), and calcium(ll) do not. Gelatin, plactoglobulin, and glutathione do not interfere, but bovine plasma albumin does, and this interference is quantitative. The copper content of several samples of ascorbic acid oxidase and tyrosinase has been determined and these data correlated with the activity and total protein content of the samples.

T

copper content of the purified enzyme ascorbic acid oxidase corresponds to 6 atoms of copper per molecule of enzyme (6). During isolation of the oxidase, the copper content becomes an index to the purity of the enzyme (12, 19). I n the case of highly purified tyrosinase the copper is directly proportional to both the cresolase (17) and the catecholase (11) activity, and hence can be used as a criterion of enzyme purity. Because the purification procedures now in use provide only very small amounts of highly purified enzyme, and these are obtained only after weeks of preparation, it is essential that a copper determination be accurate and HE

highly sensitive, so as to expend as little enzyme as possible. Depending on the reactions involved, the determination of copper in proteinaceous material may be complicated by the presence of the protein. I n such case, the protein must be destroyed by ashing. This procedure involves much time and tends to reduce the accuracy of the measurement through spattering, vaporization, transfer of solutions, etc. Colorimetric determinations of protein-bound copper have been described utilizing diethyldithiocarbamate, but these often involve ashing (6, I S ) . Micro copper determinations have been described which are not affected by extraneous protein. All of these utilize hydrochloric acid to free the bound copper. A method without any ashing requirements, based on the use of the dropping mercury electrode, determines as little as 1 y of copper per ml. with an average deviation of *3% (1). However, this procedure is timeconsuming and considerable experience is necessary before satisfactory results are obtained. The Warburg method (20) has been in use in these laboratories for some time (12). It utilizes the copper catalysis of the aerobic oxidation of cysteine to cystine, followed manometrically, and is capable of determining 0.1 y of copper in a 2.6-ml. reaction volume. Three to four hours are necessary to perform one determination in duplicate and such duplicates usually have a deviation of * 5 to 10%. Furthermore, the precision is not im-

proved significantly by using larger amounts of copper. Another method utilizing diethyldithiocarbamate has been described which does not involve ashing (3, 8). Gubler and coworkers ( 8 ) have determined as little as 0.2 to 0.4 y of copper in 3.0 ml., and given data on the precision. Peterson and Bollier (18) have described the spectrophotometric determination of serum copper with biscyclohexanoneoxalyldihydrazone, after freeing copper by the method of Gubler and coworkers (8). Gran ( 7 ) has described the use of oxalyldihydrazide for the spectrophotometric determination of low concentrations of cupric ion. This reagent, together with acetaldehyde or formaldehyde in ammoniacal solution, gives an intense blue-violet color with copper(I1). Gran reports that the molar absorbancy index for the copper(I1) - acetaldehyde - oxalyldihydrazide complex is 29,500 a t 542 mp, This unusual intensity of absorption (about three times that of the diethyldithiocarbamate-copper complex) suggests that a copper microdetermination for copper proteins, based on the use of oxalyldihydrazide, might be superior in sensitivity to the older diethyldithiocarbamate methods and easier and more precise than the Warburg method. Gran ( 7 ) has given data for solutions containing only copper and reported that the presence of reasonable amounts of hydrochloric, nitric, and perchloric acids or of sodium sulfate had no VOL 30, NO. 2, FEBRUARY 1958

191

influence on absorbance. However, he had not evaluated the effect of protein matter on the copper determination, nor the effect of other divalent metal ions, which are conimonly found in biological materials. Consequently, before the use of oxalyldihydrazide could be adapted to the determination of copper in copper oxidases, such factors had to be evaluated. MATERIALS

Water was copper-free, redistilled from distilled water in borosilicate glass, using a column equipped with a heating element to break any film (1). This water was collected in Non-Sol bottles and was used for all solutions. Standard copper solutions were made as follows: C.P. copper wire or shot was cleaned with concentrated nitric acid, washed with redistilled water, and air dried. Approximately 20 mg. were weighed on a semimicrobalance (h0.02 mg.) and dropped directly into a 1-liter volumetric flask. A few milliliters of redistilled acid were added to dissolve the copper and the solution was made to volume. Standard copper solutions, [Cu++] = 16 to 26 y per ml., were stored in copper-free Non-Sol bottles. Ascorbic acid oxidase was prepared from summer crook-necked squash as described by Dawson and Magee (4). The step involving adsorption of the enzyme to alumina gel was eliminated. Ascorbic acid oxidase samples were dialyzed exhaustively against copper-free McIlvaine's buffer, pH 5.7, before copper determinations. Tyrosinase mas prepared from the common mushroom as described by Mallette and coworkers (15). The enzyme was dialyzed exhaustively against copper-free water before copper determinations. U.S.P. gelatin was obtained from Eimer & Amend. Crystalline bovine plasma albumin was obtained from Armour & Co. The concentration of stock solutions was determined with a Beckman D U spectrophotometer a t 280 mp, E:,",. = 6.6 ( 2 ) . A sample of ,&lactoglobulin was provided by E. Brand. The concentration of stock solutions was determined spectrophotometrically a t 280 mp, E:,",, = 9.5 ( 2 ) . Glutathione was obtained from Schwarz Laboratories. Oxalyldihydrazide was synthesized by mixing diethyloxalate and hydrazine hydrate in ethyl alcohol ( 7 ) . A white precipitate separated immediately. The crude product was recrystallized from copper-free water, melting point 238.8' t o 239.2' C. with decomposition. An aqueous solution of acetaldehyde, 40%, was prepared using Eastman technical grade aldehyde. Analyzed reagent grade ammonia was obtained from J. T. Baker Chemical

co.

The hydrochloric acid was redistilled and made up t o an 0.2M solution. 192

ANALYTICAL CHEMISTRY

The bovine plasma albumin, P-lactoglobulin, glutathione, and aldehyde were used without further purification. All salts were C.P. grade and were recrystallized once from copper-free water before use. The following salts were used: cadmium sulfate octahydrate, manganese chloride tetrahydrate, nickel nitrate hexahydrate, magnesium sulfate heptahydrate, ferrous ammonium sulfate [Fe(h-H4)2(S04)2. 6H20], zinc acetate dihydrate, cobaltous sulfate (CoS04.7H20), and calcium carbonate. The carbonate was dissolved with redistilled hydrochloric acid. METHODS

All glassware was cleaned by soaking in cleaning solution, then rinsed four times each with tap water, distilled water, and copper-free water. Samples were prepared by pipetting the reagents directly into 10- or 25-ml. volumetric flasks. They mere added in the following order which Gran found most satisfactory (amounts given are for a final volume of 10 ml,): standard copper solution or copper protein, containing a total of 2 to 4 y of copper (final concentration, 0.2 to 0.4 y of copper per ml.); 1.2 ml. of concentrated ammonia; 0.8 nil. of saturated oxalyldihydrazide solution; 2.0 ml. of cold (40%) acetaldehyde solution; and copper free water to 10 ml. I n determinations with copper plus extraneous protein, protein was added after the first step. I n determinations where hydrochloric acid was used to free copper from protein, the acid was added after the first step and a t least 15 minutes before addition of ammonia. The addition of the acetaldehyde was accompanied by considerable evolution of heat. To minimize errors, the contents of the flasks were allowed to come to room temperature (about 20 minutes) before final addition of water to volume. A blank was prepared with each run, containing all reagents but copper solution (or copper protein). I n no case was more than a faint trace of color observed in this blank. I n each protein copper determination,

Table I. Oxalyldihydrazide-Acetaldehyde Copper Determination Using Lumetron Colorimeter A

c u + +,a y/M.

n. 180

Absorbance per y of Cu

0.317 0.317 0.325 0.360 0.314 0.113 0.360 0.317 0.171 0.540 0.317 0.171 0.540 0.318 0.229 0.720 0.318 0 229 0.720 0.320 0.288 0.899 0.899 0.277 0.308 Std. dev. 0.317 f 0.004 a Standard copper s o h contained 19.70 per ml. Reaction vol. was 25 ml.

0 . is0

y

Absorbance 0.057 0.057 0.117

a standard copper solution Tvas determined simultaneously and the copper content of the protein calculated from the value of the absorbance per microgram of copper given by the standard solution. This procedure was used because the absorbance value per microgram of copper of the standard solution varied slightly from day to day. However, no trend in such variation was ever observed; agreement between duplicate samples of the standard on a given day vias always excellent (see Table I). A Lumetron photoelectric colorimeter 402E (Photovolt Corp., 95 Madison Ave., New York, N. Y.) with a broad band filter and maximum transmittance a t 530 mp was used. This filter proved to be satisfactory for the measurement of the absorbancy of the complex, which had a maximum a t 542 mp, and was used in the majority of cases where no interfering colors were involved. An exception was tyrosinase, for which a narrow band filter with maximum transmittance a t 550 mp was used. This filter overlapped the 542-mp maximum of the complex and proved to be more sensitive than the 530-mp broad band filter. Hence it is recommended that a narrow band filter with a maximum transmittance a t about 550 mp be used for the measurements. Cells were cylindrical in shape; fluid volume was about 3 ml., and path width about 1 em. The reagent blank was used to zero the instrument at 100 transmission before and after each sample was read. Readings were obtained in per cent transmission and later converted to absorbance units. Dry weights of ascorbic acid oxidase and tyrosinase were determined as previously described ( I S ) . Ascorbic acid oxidase activities were determined with Warburg respirometers a t 25' C., a total volume of 10 ml., 5 mg. of ascorbic acid present in the side arm, RIcIlvaine's buffer pH 5.7, O.SJil, 5 mg. of gelatin, and air as the gas phase. A unit of ascorbic acid oxidase is defined as that amount of enzyme which will cause the initial uptake of 10 cu. mm. of oxygen per minute (4). Tyrosinase cresolase activity was determined by the method of PIIallette and D a m o n (14) and catecholase activity by the method of Miller and coworkers (16). RESULTS

During a period of several months 58 different spectrophotometric determinations, involving a number of different standard copper solutions, n ere made of the value of the molecular extinction coefficient of the copperoxalyldihydrazide complex. Absorbance readings were made a t 542 mp with two different Beckman DU spectrophotometers and gave an average value (E,) of 22,000 500 (standard deviation). The copper concentrations ranged from 0.207 to 1.114 y per ml. The value 22,000 for the molecular extinction coefficient is about 25%

*

lower than the molar absorbancy index reported by Gran (7). A 30-minute waiting period from the time the last reagent is added until the time the solutions are read on the colorimeter allows full development of color; small variations in the amounts of reagents do not affect the color intensity (7). Identical readings within experimental error viere obtained for a given copper concentration (0.206 y per ml.) in the p H range 8.2 to 10.1 ( 5 to 30yc of concentrated ammonia by volunie) . The p H dependency was not investigated below 8.2. The color intensity of the complex was constant, within experimental error, for a period of 48 hours. The data shown in Table I demonstrate that Beer's law is folloxed over the concentration range employed and indicate the degree of precision that can be achieved with this method. Cadmium(II), nickel(II), iron(II), zinc(II), cobalt(I1) gave no color with the reagents and did not interfere with the simultaneous determination of copper in concentrations approsimately equal to that of the copper. Calcium(I1) gave no color in concentrations 100 times that of the copper. Manganese(I1) gave no color with the reagents themselves, but did interfere with the simultaneous determination of copper, the effect increasing with increasing nianganese(I1) concentrations in the range \$-here the concentrations of nianganese(I1) and copper(I1) were approximately equal. Gelatin, @-lactoglobulin, and glutathione did not interfere with the simultaneous determination of copper. Some data for these substances are given in Table 11. Although the two proteins, and especially glutathione, appeared to slow the rate of color development, the final color intensity 17-as not affected. Bovine plasma albumin did interfere. Even Kith the albumin, hoTTever, the addition of 0.251 hydrochloric acid prior to ammonia prevented the interference. The effect of bovine plasma albumin TTU found to be virtually linear, as is shown in Table 111, by the constancy n-ithin experimental error, of the quantity A absorbance per milliliter of added stock solution.

Table 11.

Table 111. Effect of Bovine Plasma Albumin (BPA) and Reversal by Prior Treatment of Protein-Copper Mixture" with Hydrochloric Acid

BPA AbsorbBPAb Absorb- Added, A ancec Added, &neec Absorbance/ (0.2M M1. HC1) RI1. (Xo HC1) 0 0.205 0 0.203 1.0 0.189 -0.016 0.198 2.0 0.170 -0,018 0.197 3.0 0.158 -0.016 0.198 4.0 0.145 -0,015 0.200 Concn. of Cu++ was 0.616 y per ml. in a 25-m1. reaction vol. * BPA stock soln. contained 3.21 mg./ml. [ R L W . = 69,000 ($)I. Absorbance values are averages of duplicates. @

Table

AAO Sample 31-1

IV.

Data on Some Ascorbic Acid Oxidase (AAO) Samples"

Activity, Units/Ml. 11,900

Dry Wt. of Activity, Protein, Units/ Mg./Ml. M1. 553 21.5

Activity, Units/ of Copper, % Cu 0.123 449

Cu, r/Ml. in AAO 26.5 =k l . l b (10 detns.) 22.8 i 0 . 6 0.141 412 580 9,400 16.5 31-2, L-4 (6 detns.) 24.6 f 0.2 0.171 580 1000 31-2, L-5 14,300 14.4 (2 detns.) 31.4 f 0 . 6 0.223 645 1460 20,300 14.1 31-2, L-6 (2 detns.) 25.3 Z!Z 0 . 1 0.264 625 1650 9.6 31-2, L-7 15,800 (2 detns.) Over-all vol. for copper detns. was 10 ml. AAO 31-2 was divided into four fractions, L-4 through L-7, in increasing order of purity as measured by unitdmg. (specific activity). * Std. dev. 5

The binding of copper by bovine plasma albumin has been ascribed to the formation of a mercaptide (9). However, the interference with the copper determination cannot be explained simply in terms of S H binding, for @-lactoglobulin and glutathione also contain free SH groupings and do not interfere. Kolthoff and Willeford (10) have recently demonstrated that bovine plasma albumin forms a stable equimolar complex with copper rrhich does not involve the SH group. Specific activity data on several ascorbic acid oxidase samples and the results of copper determinations on them are presented in Table IT. The enzyme samples are arranged in order of increasing copper content, lvhich,

Effect of Proteins on Copper(l1)-Oxalyldihydrazide-Acetaldehyde Complex

Proteinaceous Material Concn., mg./ml. Gelatin 0.320 @-Lactoglobulin 0.240 Glutathione 2.79 X (No protein) ...

in the case of sample 31-2, L-7, was 0.264%. Although this copper content corresponds t o that of the pure enzyme, the specific activity data on this sample indicate that it was about 83% pure. Pure enzyme has a specific activity of 2000 units per mg. (6). I n all of the determinations on ascorbic acid oxidase, the precision of measurement was satisfactory. When 0.2J1 hydrochloric acid was added to these partially purified specimens of ascorbic acid oxidase, no precipitation of protein and no turbidity in the solution m-as noted. Furthermore, the solutions were essentially colorless prior to the final addition of acetaldehyde. Data for a highly purified tyrosinase

Cu

Added,a y/Ml. 0.616 0.616 0.616 0.616

+

+

Absorbance (Av. of Duplicates) 0.206 0.204 0.205 0.205

Each 25-ml. reaction vol. contained constant amt. of oxalyldihydrazide and acetaldehyde.

preparation and the results of copper determinations on it are presented in Table V. When hydrochloric acid was added t o the tyrosinase, significant turbidity was observed. Consequently the solutions were centrifuged clear, after addition of all reagents, before being read. The data given in Table T' have been arranged to show that copper was not coprecipitated-Le., all of the copper of the enzyme vas available to the oxalyldihydrazide reagent. This iact was established by adding known amounts of copper(I1) to the enzyme specimen in four separate experiments and noting that in each case the total copper in the system, as determined by the oxalyldihydrazide reagent, was the sum of the previously determined copper in tyrosinase and the added copper, T, ithin experimental error. Highly purified tyrosinase specimens did not give sufficient color in the determination to warrant correction Khen a narrow band filter n-as used. Cruder preparations are highly colored (yellow to bronTn) and are sometimes quite turbid. When this type of preparation was encountered, the follo1Ting modifications viere employed: The tyrosinase and the 2.OM hydroVOL. 30, NO. 2, FEBRUARY 1958

* 193

Table V.

Copper Determinations on Tyrosinase“

Tyrosinase, M1.

Added CU, -y/Ml.

0.08 0.08

... ...

Absorbance 0.065 0.067

0.08

0.257 0.257

0.174 0.175

0.08

0.10 0.10

.

I

.

. I .

0,082

0.085

Total Cu, 7/Ml. 0.158 0.163

Tyrosinase Cu, y/ML 0.158 0.163

0.432 0.425 0.207 0.214

0.166

Cu in Original Tyrosinase Prepn., y/ML 20.4

Recovery of added copper with this type of preparation was satisfactory. DISCUSSION

The oxalyldihydrazide-acetaldehydeammonia method for the microdetermination of copper is extremely simple xhen applied to proteins from which copper can be freed by simple acid treatment and Tvould be complicated only slightly if a wet digestion were necessary to free the copper. Many samples can be analyzed simultaneously with rapidity and ease. As little as 0.1 y of copper per ml. can be determined with a precision to = t 2 to 3%. Even smaller concentrations might be determined by increasing the path length, a simple matter with

LITERATURE CITED

=k 0 . 6

0. is8

0.207 0.214

21.6 i 0 . 8 0.10 0.161 0.150 0.378 0.217 0.10 0.161 0.147 0.370 0.209 Over-all vol. for copper detns. was 10 ml. Dry wt. of tyrosinase prepn. was 12.5 mg./ml. Catecholase and cresolase activity were 35,000 units/ml. f2800 units/mg.) and 500 units/ml. (40 units/mg.), respectively. chloric acid (not 0.21M) were pipetted accurately into a centrifuge tube, stirred Tell, and centrifuged. Aliquots of the clear but highly colored supernatant were used in the determination. A blank for tyrosinase color was prepared with all reagents except oxalyldihydrazide and was read against distilled water. The absorbance of this blank was then subtracted from the optical density given in the copper determination to give optical density due t o copper.

in the p H range 8.2 to 10.1). The ease of procedure, time necessary for determination, number of samples which can be determined simultaneously, and precision are comparable for the two methods.

the Lumetron, which has a large and adaptable cell compartment. Because of their similarity, it seems pertinent to compare the biscyclohexanoneoxalyldihydrazone method of Peterson and Bollier (18) with the oxalyldihydraxide-acetaldehyde method presented here. One of the most important differences is that the acetaldehyde complex is generated in situ, whereas the cyclohexanone hydrazonc is pre-formed. The new method involving acetaldehyde is more sensitive. than the method involving biscyclohexanoneoxalyldihydrazone, because the molecular extinction of the copper complex formed with acetaldehyde is 22,000 as compared with 16,000 for the cyclohexanone. I n the latter case. the color intensity is stable up to 1 hour and then fades a t the rate of approximately 1% per hour, whereas the color intensity of the acetaldehyde complex is stable for at least 48 hours. The acetaldehyde complex is less sensitive to p H changes. This is demonstrated by the fact that Peterson and Bollier found it necessary to titrate to the phenolphthalein end point (pH 7.5 to 7.9) whereas such titration is not necessary with the acetaldehyde method (color constant and maximum

(1) Ames, S., Dawson, C. R., IND. ENG.

CHEM.,ANAL.ED. 17, 249 (1945). ( 2 ) Boyer, P.D., J . Am. Chem. SOC. 76, 4331 (1954). (3) Chatagnon, C., Chatagnon, P., Bull. SOC. chim. biol. 36, 911 (1954).

(4) Dawson, C. R. Magee, R. J., “Methods of hnxyrnology,” Vol. 11, p. 831, Academic Press, Kew York, 1964. (5) Dunn, F., Dawson, C. R., J . Bzol. Chem. 189, 485 (1951). ( 6 ) Eden, A., Green, H. W., Biochern. J . 3 4 , 1202 (1940). ( 7 ) Gran, G Anal. Chim. Acta 14, 150 (l$66). (8) Gubler, C. J., Lahey, M. E., Ashenbrucker, H., Cartwright, G. E., Wintrobe, M. M.,J . Bzol. Chem. 196, 209 (1952). ( 0 ) Klotz, I. M.,Urquhart, J. M., J . Am. Chem. SOC.74, 5637 (1952). ( l a ) Kolthoff, I. M., Willeford, B. R., Jr., Ibid., 79, 2656 (1957). (11) Kubowitz, F., Biochem. 2. 292, 221 (1937); 299, 32 (1938). (12) Lovett-Janison, P., Nelson, J. RI., J . Am. Chmn. SOC. 62, 1409 (1940). (13) RIcFarlane, W. D., Biochem. J . 26, 1022 (1932). (14) Mallette, M. F., Dawson, C. R., J . Ani. Chmn. SOC.69, 466 (1947). (15) Mallette, M. F., Lewis, S., Amea, S. R., Nelson, J. M., Dawson, C. R., Arch. Biochem. 16, 283 i1948). (16) XI;ller,-’iV. H., Mallette, M. F., Roth, L. J., Dawson, C. R., J . Am. Chenz. SOC.66, 514 (1944). (17) Parkinson, G. G., Nelson, J. RI., Ibid.,62, 1693 (1940). (18) Peterson, R. E., Bollier, M., ANAL. CHERT. 27, 1195 (1955). (19) Powers, W. H., Lewis, S., Dawson, C. R., J . Gen. Physiol. 27, 167 (1944). (20) Warburg, O., Krebs, H. A., Riochem. Z. 190, 143 (1927). RECEIVEDfor review July 5, 1057. AlcceptedOctober 3, 1957.

X-Ray Powder Diffraction Data of Several Cobalt Ammine Azides TAYLOR B. JOYNER, DONALD S. STEWART, and LOHR A. BURKARDT Chemistry Division, Research Department, ,During a study of some physical properties of cobalt ammine azides, x-ray diffraction patterns were taken of each preparation. Some of the diffraction data accumulated in this manner are presented here.

194

ANALYTICAL CHEMISTRY

U. S.

Naval Ordnance Test Station, China lake, Calif.

D

studies of the physical properties of cobalt ammine azides, an x-ray diffraction pattern was taken of each powder preparation as soon as it was made. The x-ray patterns are a simple means of characterizing the URIXG

preparations. I t was hoped that these patterns Iiiight ax-oid difficulties encountered in the study of some related cobalt compounds, when materials of similar prepration slioTved markedly different phj-eical properties.