to dehydrate sample 1015 and to stop heating before the start of the exotherm. X-ray diffraction of the dehydrated material heated to 300" C. gives a completely amorphous pattern, After the same sample is heated t o 600' C., a strong hematite pattern is found. The conversion peak is always a doublet, suggesting that it takes place in two steps. The exotherm may be attributed, therefore, to the conversion of the lepidocrocite defect structure or of amorphous ferric oxide to hematite. Lepidocrocite reduced by hydrogen at 400' C. and reoxidized a t room temperature shows two exotherms upon heating in air: one a t 400' C. and the other a t 725" C. The combined areas under these exotherms are of the same magnitude as that of the y- to a- conversion of goethite. Heat of y- to a- Conversion of FezOa. The heat of the y- to CY- conversion of FezO, can be determined from the area under the high-temperature exotherm given in Table 11. Integrated area under peak for 400 mg. of goethite: 4.5 =k 0.1 sq. em.
Sensitivity of thermal analysis between 700" and 900" C. (from calibration with CaCOJ: 3.8 i 0.1 cal. per sq. cm. 3.8 X 4.5 = 171.1 & 0.83 cal. for 360 mg. FenOs
Weissman and his associates of the Bureau of Engineering Research of Rutgers, who also graciously permitted the use of their x-ray diffraction facilities. REFERENCES
or 47.5
2.3 cal. per gram
Similarly, the heats of other transformations can be calculated from the peak areas. Effect of Hematite i n Raw Sample. Heating in hydrogen t o 450' or 500' C. during DTA reduces hematite particles only at t h e surface. Because even finely ground hematite has very little surface area compared to that of dehydrated iron hydroxides, the effect of hematite present in the raw samples on the size of the y- to a- conversion peak is negligible. ACKNOWLEDGMENT
Valuable advice was given by Sigmund
( 1 ) Bernal, J. D., et al., Clay Minerals Bull. 4 (21), 15-30 (1959). (2) Francombe, M. H., Rooksby, H.P., Ibad., 4 (21), 1-14 (1959). (3) Kopp, G. C., Kerr, P. F., Am. Mineralogist 42, 445-54 (1957). (4) Kulp, J. L., Kerr, P. F., Ibid., 36, 2344 (1951). (5) Lodding, W., Hammell, L., Rev. S C ~In&. . 30, 10 885-6 (1959). (6) Mackenaie, R. b., "Problems of Clay and Laterite Genesis," Am. Inst. Min. and Met. Engrs., 1952. (7) Paulik, F., Erdey, L., Acta Chim. Acad. Sci. Hung. 13, 117-39 (1957). (8) Wells, A;, F., "Structural Inorganic
Chemistry, Oxford Univ. Press, Oxford, 1950.
RECEIVED for review August 31, 1959. Accepted December 28, 1959. Published by permission of Helgi Johnson, Director of the Bureau of Mineral Research, R u b pers, The State University.
A n a Iys is of Bis muth-Ant imo nyTe I I urium- Se Ie nium Co mbina t io ns JAMES F. REED Technology Depaffmenf, Westinghouse Research Laboratories, Pittsburgh 35, Pa. In the analysis of mixtures containing bismuth, antimony, selenium, and tellurium each element interferes with the determination of one or more of the others. However, suitable separations are possible. Selenium and tellurium are separated at different acidities with sulfur dioxide and weighed as the free elements. Bismuth is titrated with (ethylenedinitrilo]tetraacetic acid with thiourea as the indicator. Antimony is titrated potentiometrically with potassium permanganate. Accuracy and precision are within 1 to 2 parts per thousand.
T
HE analysis of combinations of bismuth, antimony, selenium, and tellurium is useful for the investigation of their thermoelectric properties. The determination of any one of these elements is straightforward, but there are mutual interferences and other intrinsic difficulties. For example, selenium and tellurium interfere in the permanganate titration of antimony. Antimony interferes by hydrolysis in
662
ANALYTICAL CHEMISTRY
the titration of bismuth with (ethylenedinitrilo) tetraacetic acid (ethylenediaminetetraacetic acid, EDTA), and tellurium reacts with the indicator, thiourea, The reducing agents used for the separation of selenium and tellurium must be removed before antimony can be titrated. Finally each element except bismuth forms a volatile Chloride, yet hydrochloric acid is required to maintain all four elements in solution. Hillebrand and coworkers (4) separated selenium and tellurium at different hydrochloric acid concentrations with sulfurous acid, and dried both elements in air a t 110' C. Duval(1) showed that tellurium oxidizes a t temperatures above 40' C. This work shows that tellurium may be successfully dried in vacuo at room temperature. Fritz (8) titrated bismuth with EDTA using thiourea as a n indicator at a p H of 2.0. Gronkvist (3) recommended a pH range of 2.5 to 4.0 and a higher concentration of thiourea, In this work a p H of 2.4 is preferred when the titration is carried out in the presence of antimony. Also,
with antimony present, values are erratic above p H 2.8. Potentiometric titration of antimony with permanganate eliminated the difficulties associated with ice baths and fading visual end points. EXPERIMENTAL
Reagents. Sulfurous acid, saturated solution of sulfur dioxide in water. Hydrazine hydrochloride, 15% (w./w.) in water. Thiourea. (Ethylenedinitri1o)tetraacetic acid (EDTA), disodium salt, 0.01M in water, standardized against pure bismuth. Potassium permanganate, 0.01 or 0.05N, standardized against sodium oxalate. Acid sulfide wash solution, 1.1N sulfuric acid saturated with hydrogen sulfide. Apparatus. Fisher Titrimeter o r equivalent with calomel platinum electrodes. Procedures. DETERMINATIONOF SELENIUM. Dissolve about 0 . 5 gram of sample in 10 ml. of nitric acid and evaporate t o dryness. Dissolve t h e residue in 100 ml. of concentrated
-
hydrochloric acid. Add 25 ml. of E D T A complex as compared to the sulfurous acid. Stir and set aside at Table I. Effect of pH on Bismuth" thiourea complex. Erratic values room temperature for 3 hours. Filter Determinations were obtained a t higher p H because through a weighed fritted crucible. the color of the indicator complex was Bi Wash the selenium in the beaker once Found, too weak for easy visual titration. This with concentrated hydrochloric acid. Gram PH is due to the presence of tartrate, Transfer the selenium to the crucible 1.6 0 0458 necessary to complex antimony, which with 0.2N hydrochloric acid. Then 1.6 0.0445 competes with the thiourea in complexwash the crucible twice with methanol. 2.0 0.0420 Dry the selenium for 1 hour a t 105' C. ing bismuth above a p H of 2.8. Stand2.0 0.0421 Cool in a desiccator and weigh as the ardization of EDTA against bismuth 2.0 0,0420 free element. 2.2 a t the same pH is recommended. 0.0418 DETERMIXATION OF TELLURIUM. To 2.4 0.0418 Interferences. As pure materials the filtrate from the selenium deter2.7 0.0418 were used in the preparation of the mination add 2 grams of tartaric acid to a 0.0418gram taken. thermoelectric materials, there was keep the antimony from hydrolyzing. little interference. Even if present, Add water until the hydrochloric acid only gold would interfere with the concentration is 3M. Add 10 ml. Table 11. Analysis of Known Mixtures selenium and tellurium determinaof hydrazine hydrochloride solution and another 25 ml. of sulfurous acid. Boil tions. Other reducible metals such Taken, Found, Error, 1 or 2 minutes. Filter through a fritted as iron and vanadium interfere in the Mg. Mg. Mg. crucible. Transfer the tellurium to the titration of antimony, but the sulfide Bismuth crucible with 0.2N hydrochloric acid. separation of antimony removes most Then wash the crucible with 0.2N 51.2 51.3 $0.1 of these. Of the common metals, only 103.7 103.7 0 hydrochloric acid, and finally with iron and zirconium are titrated with 104.1 103.9 -0.2 water. Dry the precipitate by placing 104.1 104.1 0 EDTA in the acid solution recomthe crucible and tellurium in a desic$0.2 119.8 120.0 mended for bismuth. Antimony does cator a t 20-mm. pressure or less a t room -0.2 216.8 216.6 not interfere if it is complexed by tartemperature over anhydrous calcium sulfate for 2 hours. Weigh a s the free Antimony trate and the pH is controlled. element. Accuracy and Precision. Typical 3.90 3.87 -0.03 DETERMINATIONOF ANTIMONY. 0 results for each element are given in 7.81 7.81 Place the filtrate from the tellurium 11.71 11.71 0 Table 11. Synthetic samples, made determination in a volumetric flask 19.49 19.54 +0.05 from appropriate amounts of each 52.2 52.9 $0.7 and divide into two parts, one for antielement, were carried through the -0.2 70.5 70.3 mony and one for bismuth. Dilute the entire procedure. aliquot for antimony so that the hydroSelenium The procedure may be shortened chloric acid concentration is IX. Pre59.5 $0.1 59.4 by determining one element, such as cipitate the antimony (and bismuth) 118.2 118.3 -0.1 with hydrogen sulfide. When the prebismuth, by difference. Table I11 119.9 120.1 -0.2 cipitate has settled, filter through shows t h a t such a determination is 120.6 120.6 0 paper. Wash with 1.1N sulfuric acid 131.7 satisfactory if no large amounts of 131.4 +0.3 saturated with hydrogen sulfide. Trans137.1 137.4 -0.3 other elements are present. fer the paper and precipitate to the 140.1 139.7 +0.4 The data in Tables I1 and I11 were -0.2 180.6 180.8 same beaker. Add 20 ml. of sulfuric analyzed statistically by methods deacid and enough nitric acid to destroy Tellurium scribed by Mood ( 5 ) or Youden (6) organic matter. Bring to fumes of 20.0 (Table IV). In this type of statistical 19.9 $0.1 sulfuric acid to remove the excess nitric 59.5 0 59.4 consideration the data are fitted to the acid. Dilute with 2 parts of water, and 189.2 -0.1 189 3 add 20 ml. of sulfurous acid. By vigorequation 190.3 0 190.3 ous boiling, reduce the volume by half 195.0 -0.3 195.3 to remove the excess sulfur dioxide. $0. 1 196.5 196.4 y=a+bt Dilute to 200 ml. and add 20 ml. of +O.l 203.5 203.4 hydrochloric acid. Titrate the anti$0. 1 205.9 205.8 mony potentiometrically with potassium where 2/ is the amount found, x is the permanganate. The valence of the antimony changes from 3 to 5. DETERMINATION OF BISMUTH. EvapTable 111. Bismuth by Difference orate the aliquot for bismuth to dryness. Redissolve in a minimum of 1 N nitric Added, Mg. Found, Mg. Error, Mg. By Diff., Mg. Error, Mg. acid, addin more tartaric acid if 48.2 48.4 $0.2 48.2 0 necessary. ilute to 60 ml. and add 37.1 36.9 -0.2 -0.4 36.7 1 gram of thiourea. Adjust the p H to 28.8 28.6 -0.1 -0.2 28.9 2.4 =k 0.1 with ammonia. Titrate with 19.7 19.7 0 19,6 -0.1 EDTA until the yellow color of the 11.0 11.1 $0.1 11.2 +0.2 bismuth-thiourea complex just disappears. One milliliter of 0.01M EDTA is equivalent to 2.09 mg. of bismuth, Table IV. Statistical Data 95% Confidence Intervals Std. dev., RESULTS AND DISCUSSION Identification a,mg. b a b mg. Bi 0.168 0,998 -0.32, +0.65 0.994, 1.002 0.2 Importance of pH. This effect was Sb -0.020 1.002 -0.60, +0.56 0.956, 1,048 0.3 Se 0.191 0.998 -0.82, +l.ZO 0,990, 1.006 0.3 investigated with known amounts of Te 0.062 1.000 -0.26, $0.38 0.2 0.998, 1.002 bismuth in the presence of antimony (Table I). Results are fairly conTable 111, Bi sistent from p H 2.0 t o 2.7. Results a t Direct -0.036 1.001 -0.75, $0.68 0.978, 1.023 0.2 lower p H were high because of the 0.164 By difference 0.993 -0.65, +0.98 0.911, 1.075 0.2 weaker strength of the bismuthI
I
!6
VOL. 32,
NO. 6, MAY 1960
663
amount taken, and a and b are the intercept and slope, respectively. In all cases the confidence interval for the slope includes unity, indicating that the analyses are accurate and consistent over the range covered. Also the confidence interval for the intercept includes zero, from which i t is inferred that in no case is there a blank dependent on the amount of constituent. The estimated standard deviation for bismuth difference would be expected to be somewhat larger than
reported here, because it should contain the errors Of the Other determinations. The fact that it is not larger suggests the presence of compensating errors. However, because the precision was satisfactory, no effort was made to isolate these errors.
(3) Grbnkvist, K. E., Farm. Roy. 52, 305 (1953). (4) Hillebrand, W. F., Lundell, G. E. F., Bright, H, A,, Hoffman, J. I., L‘Applled Inorganic Anal ais,” pp. 280, 334, Wiley, New Yorc, 1953. (5)Theory ofA*Statistics, M., “In!foduction Chaps. 11, to the 13, McGraw-Hill, New York, 1950.
LITERATURE CITED
York, 1951. RECEIVED for review October 19, 1959. Acce ted January 22, 1960. Pittsburgh Conzrence on Analytical Chemistry and A plied Spectroscopy, Pittsburgh, Pa., $arch 1959.
(1) Duval, Clement, “Inorganic Thermoravimetric Analysis,” pp. 302, 384, New York, 1953 (2) Fritz, J. S., ANAL. CHEM. 26, 1978 (1954).
blsevier,
(6) Youden,, W.,,J., “Statistical Methods for Chemists, Chap. 5, Wiley, Kew
Automated System for Continuous Determination of Penicillin in Fermentation Media Using Hydroxyla mine Reagent A.
0.NIEDERMAYER, F. M. RUSSO-ALESI, C. A. LENDZIAN, and JACQUES M. KELLY’
Squibb lnstitufe for Medical Research, New Brunswick, N. .I.
b An automated colorimetric method for the determination of penicillin based on the reaction of penicillin with hydroxylamine and ferric ion is presented. It is valid in the range of 0 to 10,000 units per ml. and compares satisfactorily with the microbiological diffusion assay. The method has been used extensively in the control of pilot and plant fermentations. The standard error is 2.5%.
A
x AUTOMATED METHOD for the chemical determination of penicillin in fermentation media using the Technicon AutoAnalyzer has been described (4). The chemistry of the method is based on the inactivation of penicillin to penicilloic acid, either enzymatically or chemically (1). The preformed penicilloic acid is separated by means of the continuous dialysis unit of the AutoAnalyzer. Potencies are estimated by colorimetrically measuring the differences in iodine uptake, between active and degraded penicillin. I n initial trials, the method was reasonably satisfactory in most cases. However, its limitations became evident as broader concentration ranges and widely varying fermentation media were studied. Valid analyses of many fermentation media encountered were confined to rather narrow ranges of 1 Present address, Charles Pfizer & Co., Brooklyn, N. Y.
664
ANALYTICAL CHEMISTRY
concentration and necessitated the preparation of aqueous penicillin standards a t levels approximating those of the samples. The best workable concentration range was of the order of magnitude of 400 to 1600 units per ml. Outside this range, pronounced curvature was , observed and, hence, the samples required dilution and reanalysis. Furthermore, with some fermentation media, the responses of serially diluted samples with water were not linear. The reason for this phenomenon is still not clearly understood. However, it can be demonstrated that the dialysis of serially diluted aqueous penicillin standards through the membrane is a curvilinear function. Details of this work will be reported elsewhere. Attempts to use the iodine method without dialysis were unsatisfactory because of gross interferences from broth constituents. Hydroxylamine was first suggested as a reagent for penicillin by Boxer and Everett (8). Penicillin reacts with this reagent and ferric ion to give a colored complex. On the other hand, its alkaline or penicillinase degradation product does not give a colored complex. Therefore, by running a sample and blank determination, the penicillin potency can be estimated from the difference in the color response. The method has been widely used in the fermentation industries. The details of this method have been modified for its automated operation on the AutoAnalyzer.
METHOD OF ANALYSIS
A standard type Technicon AutoAnalyzer is used without the continuous dialyzer. One additional time-delay coil is used in the system to permit adequate inactivation of penicillin by penicillinase. The coil is a glass helix of sufficient length to give a 5-minute contact time. Reagents. PENICILLIN STANDARDS. Dissolve a predetermined weight of potassium benzylpenicillin in sufficient distilled water to give the equivalent of 10,000 units per ml. Dilute this stock standard with distilled water t o give standard solutions of 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, and 10,000 units per ml. Use these levels of concentration t o construct the standard curve. PENICILLINASE. Dissolve 100,000 units of Schenley penicillinase A in 150 ml. of distilled water a t room temperature. Store the reagent in an ice water bath. It is stable under these conditionu for a t least 8 hours. HYDROXYLAMINE REAGENT(STOCK SOLUTION).Dissolve 350 grams of “*OH. HC1 (J. T. Baker) in water and dilute to 1 liter. The reagent is stable for a t least 2 weeks a t room temperature. ALKALIBUFFER (STOCKSOLUTION). Dissolve 173 grams of sodium hydroxide and 20.6 grams of anhydrous sodium acetate in water and dilute to 1 liter. WORKING HYDROXYLAMINE REAGENT. Titrate a portion of the alkali buffer solution against the hydroxylamine hydrochloride solution t o p H 7.0, using a pH meter. To 1 volume of neutralized NHzOH. HC1 solution add 8 volumes of water and 2 volumes of 95y0 ethyl alcohol. This reagent is
