1192
ANALYTICAL CHEMISTRY
Table V.
Simultaneous Determination of Cyanate and Cyanide Found, P.P.M. OCN 0.22 0.40 1.04 2.01 5.13 10.0
C ?i 0.36 0.40 0.44 0.40 0.42 0.38
oc s
0.34 0.44 1.17 2.09 5.02 9.84
0 30 0.42 0.38 0 40 0 42 0.42
0.20 0.40 1.00 2.00 5.00 10 0 0.20 0.40 1.00 2.00 5.00 10.0 0.20 0.40 1.00 2.00 5.00 10.0
0.23 0.46 1.00 2.05 4.75 10.0 0.27 0.41 0.98 1.83 5.06 9.55 1.30 1.20 1 92 3 32 5 83 8 78
1.00 0.75 1.08 1.04 1,02 1.06
0.25 0.36 1.02 2.08 4.88 10.0
1.04 1.00 1.02 0.96 1.08 1.13
1.93 1.74 2.00
0.36 0.54 1.06 2.10 4.94 9.28
1.89 2.00 2.08 2.00 1.96 1.98
1.00 1.00 1.00 1.00 1.00 1.00 2.00 2.00 2.00 2.00 2.00 2 00 10.0 10.0 10.0 10.0 10.0 10.0
Trial 1
Trial 2
Present, P.P.M. OCN CN 0.20 0.40 0.40 0.40 1.00 0.40 0.40 2.00 5,oo 0.40 IO. 0 0.40
1.95
2.00 2.03 9.4 11.0 IO. 5 12 3 12 3 9 3
0.88 1.30 1.35 2.18 4.55 7.55
CT
10.9 12.0 11.8 12.2 11.6 11.1
of cyanate and cyanide containing more than 0.2 p.p.ni. of cyanide. For the analysis of unknown samples, proceed as described for ammonia. Always make the extraction with 50 ml. of carbon tetrachloride 150 seconds after the addition of the reagent. Measure the absorbance of the extract a t 450 and 580 mp, and subtract the absorbance of the reagent blank. From the absorbance a t 580 mp, determine the concentration of cyanide and calculate the absorbance a t 450 mp due to cyanide by using the
ratio of absorptivities found for the 0.5 p.p.m. cyanide solution. Subtract this absorbance from the total absorbance a t 450 mM; the residual absorbance is due to cyanate. From the cyanate calibration curve it is then possible to obtain the amount of cyanate present. The above procedure and method of calculation were used for various mixtures of cyanate and cyanide (Table V). The method was found to be rapid, so that a set of nine solutions could be tested in 45 minutes. hCKNOWLEDG.MENT
The authors gratefully acknowledge the support of this work through a grant by The Xational Institutes of Health, United States Public Health Service. LITERATURE CITED (1) Bailey, K. C., and Bailey, D. F., J . Sac. Chem. I n d . , 44, B 313 (1926). (2) Bougault, J.. arid Gros, R., J . pharm. chim., 26, 5 (1922). (3) Dodge, B. F., and Zabban, W., Plating, 3 9 , 3 8 1 (1952). (4) Geiger, E., Hela. Chim. Acta, 25, 1453 (1942). (5) Jenkins, 8. H., Inst. Sewage Purif., J . and Proc., 1950, 147. ( 6 ) Kolthoff, I. AI., Pharin. Weekblad, 57, 1253 (1920). (7) Kruse, J. AI., with hIellon, M. G., Sewage and Ind. Wastes, 24, 1264 (1952). (8) Kuisel, H. F., Helv. Chim. Acta, 18, 186 (1935). (9) Kulenok, AI. I., Gigiena i Sanit., 1950, No. 10, 45. (10) Reuss, A., 2. Untersuch. Lebensm., 73, 50 (1937). (11) Thompson, J. F., and Morrison, G. R., A N ~ LCHEW, . 23, 1153 (1951). (12) Werner, E. A , , J . Chem. SOC.,123, 2577 (1923).
RECEIVED for review December 9, 1952. Accepted M a y 25, 1953. Based in part on a thesis presented by J. M. Kruse t o Purdue University in partial folfillnirnt of the requirements for the Ph.D. degree.
Colorimetric Determination of Calcium Pantothenate C. R. SZALKOWSKI AND J. H. DAVIDSON, JR. Control Division, Merck & Co., Inc., Rahway, N . J .
C
ALCIUM pantothenate is a member of the vitamin B complex vitamins and is used extensively as a constituent of multiple vitamin formulations and a variety of animal food supplements. Therefore, a rapid and accurate chemical method for its determination is highly desirable. Up to the present, control of this constituent in these mixtures has been based on microbiological assays ( 2 , 7 ) . Chemical methods using 1,2naphthaquinone-4-sodium sulfonate ( 1) and 3,4-dinitrophenylhydrazine ( 5 ) have been described for the determination of p-alanine, a hydrolytic cleavage product of pantothenic acid, A colorimetric method (8) has been reported based on Feigl’s ( 3 ) reaction for esters and lactones after hydrolysis of pantothenic acid or panthenol to pantoyl lactone.
lowed over a suitable concentration range, with either acid or alkaline hydrolysis. The specificity of this reaction with respect to other vitamins organic and amino acids, and certain diluents usually encountered in vitamin mixtures has been studied. The presence of relatively large amounts of such compounds as thiamine, pyridoxine, choline, niacin, niacinamide, vitamins h and D, vitamin BIZ, atocopherol, citric and tartaric acids, and palanine do not interfere with the pantothenate estimation. Such compounds as riboflavin, ascorbic acid, lactose, glucose, gluconic acid, glycolic acid, and furfural, however, do interfere with the reaction. Means of removing these interfering substances are given. REAGENTS
PRINCIPLE OF METHOD
Hydrolytic cleavage of pantothenic acid in acid medium results in the formation of p-alanine and a,r-dihydroxy-P,P-dimethylbutyric acid. In an acid medium a t a pH below 5, the dihydroxy acid undergoes Iactonization to form a-hydroxyp,@-dimethylbutyrolactone( 4 ) . Hydrolysis in alkaline solution will result in the formation of a,y-dihydroxy-p,P-dimethylbutyric acid. The dihydroxy acid or the lactone produced by the hydrolysis reacts with 2,7-naphthalenediol in concentrated sulfuric acid to form a greenish yellow colored complex. The ratio between the amount of the colored complex formed and the hydrolyzed pantothenic acid is constant and can be estimated in dilute sulfuric acid spectrophotometrically a t 465 mp. Beer’s law is fol-
Unless otherwise indicated, all reagents are C.P. or reagent grade. Sulfuric acid. Cupric sulfate, anhydrous. Calcium hydroxide. Diluted sulfuric acid. Mix 100 ml. of distilled water with 100 ml. of concentrated sulfuric acid. Cool in ice bath. Naphthalenediol reagent. Dissolve 500 mg. of 2,i-naphthalenediol (Eastman) in 500 ml. of concentrated sulfuric acid. Allow this solution to stand until practically colorless (about 18 to 24 hours). Keep protected from light and prepare fresh weekly. Acetic acid-pyridine solution. This is a mixture prepared with distilled water containing 207, of pyridine and 2% of acetic acid (v./v.). Florisil. To a 500-ml. beaker add 100 to 200 grams of 60- to
V O L U M E 2 5 , NO. 8, A U G U S T 1 9 5 3 Calcium pantothenate, a member of the vitamin B complex vitamins, is used extensively as a constituent of multiple vitamin formulations and a variety of animal food supplements. Therefore, it is highly desirable that there be a rapid and accurate chemical method for its determination. By use of a Florisil column and a cupric sulfate-calcium hydroxide treatment for the adsorption and elimination of interfering vitamins, the method is rendered specific for calcium pantothenate. The method is based on the formation of a,y-dihydroxy-P,P-dimethylbutyric
100-mesh Florisil (Floridin Co., Warren, Pa.) and cover with sufficient acetic acid-pyridine solution, boil gently with constant stirring for several minutes, then allow t o settle. Decant the supernatant liquid and repeat the washing and decanting two times; finally wash the Florisil thoroughly with hot distilled water to remove pyridine and acetic acid. Dry the prepared Florisil in an oven a t 100" to 120" C. Store in a closed container. Standard calcium pantothenate. Weigh 100.0 mg. of d-calcium pantothenate, United States Pharmacopoeia reference standard, dried a t 105" C. for 3 hours, into a 100-ml. volumetric flask, dissolve in distilled water, dilute to 100 ml. with water, and mix. Each milliliter contains 1.0 mg. of calcium pantothenate. Florisil column. Use a glass column 12 mm. in inside diameter, approximately 50 to 75 cm. in length, and constricted a t the lower end. Place a plug of glass wool in the constricted end and slowly add 4 grams of prepared Florisil, applying gentle suction. Kash the column with 25 ml. of distilled water and use.
1193 acid by hydrolytic cleavage in acid medium. The hydroxy acid reacts with 2,7-naphthalenediol in presence of sulfuric acid to yield a colored complex. This color, with a maximum absorption at 465 mr, is utilized for the colorimetric determination. The effects of hydrolysis, temperature, and quantities of reagents added are described and data concerning the stability of the color and precision of the method are given. This new method shows very good agreement with the recognized microbiological method and greater precision and accuracy.
a t the temperature of a boiling water bath. The temperature effect is shown in Table 11. For best reproducibility the reaction is carried out for 30 minutes in a boiling water bath, in view of the fact that the standard of about the same concentration as the sample is run simultaneously and this is subjected to exactly the same conditions. Effect of Naphthalenediol. To determine the effect of various quantities of naphthalenrdiol, experiments were run using 10 ml.
RECOMMENDED PROCEDURE
Keigh 100.0 mg. of the sample of calcium pantothenate into a 100-ml. volumetric flask, dissolve in distilled water, dilute to the 100-ml. mark with water, and mix well. Measure 25.0-ml. aliquots of the sample solution into 250-ml. standard taper flasks, add 40 ml. of water and 5 ml. of sulfuric acid. Connect to a reflux condenser and boil gently for 1 hour. Cool to room temperature, quantitatively transfer the solution to a 100-ml. volumetric flask, and dilute to the mark with water. Measure 1.0-ml. aliquots of the h drolyzed solution into 2.5. X 20 cm., glass-stoppered tubes (the yength of the tubes is an important factor in preventing water vapors from diluting the sulfuric acid during the reaction in the boiling mater bath). Place these tubes in an ice bath for 5 minutes. Then from a pipet or buret add 10.0 ml. of the naphthalenediol reagent to each tube. Make the mixtures homogeneous by carefully shaking the tubes. Place the tubes in a boiling water bath for 30 minutes. Remove the tubes from the bath and cool them in an ice bath for 5 minutes, then add from a pipet or buret 15.0 ml. of diluted sulfuric acid. Mix thoroughly and allow to stand a t room temperature for 30 minutes. Read the color in a spectrophotometer set a t 465 mfi against a blank prepared at the same time from the reagents. Preparation of Standard. Measure 25.0 ml. of the standard calcium pantothenate solution into a 250-ml. standard taper flask. Add 40 ml. of water and 5.0 ml. of sulfuric acid, connect to a reflux condenser, and proceed in exactly the same manner and at the same time as the sample. EXPERIMEi'TAL
The procedures described were arrived a t after investigation of all factors which have a bearing upon the developnient of the final color. Hydrolysis. Table I shows that unhydrolyxed pantothenate yields the color when reacted with the naphthalenediol reagent, but the reproducibility of the color formation is poor. Hydrolysis of the pantothenate produces a threefold increase in the color formation and improves the reproducibility. The acid or alkaline hydrolysis of the pantothenate goes to completion in 1 hour. Effect of Temperature. Hydrolyzed pantothenic acid produces very little color with the naphthalenediol reagent a t room temperature. Increase in temperature causes an increase in color formation. Maximum color is developed in 30 to 60 minutes
I
I
I
I
I 6W
5 00
400 MU
Figure 1. Absorption Curves a,-pDihydrox, -p,@-dimethylbut>-rolactone Calcium pantothenate 10-mm. cell; 20 micrograms per milliliter
A. B.
Table I.
Absorbancies Produced by 250 Micrograms of Calcium Pantothenate Unhydrolyzed
Table 11.
Hydrolyzed
Effect of Temperature on Color Formation
(250 micrograms of calcium pantothenate; time 30 minutes) Temp., C. Absorbancy 20-250 0.01 40 0 05 60 0 07 100 (boiling water b a t h ) 0 550
1194
ANALYTICAL CHEMISTRY
Sample
Table 111. Reproducibility of Results CaPan, % ’ Average, 7% Std. Deviation, “c 100.0, 99.6, 100.2, 99.2 98.5,97.4,99.3,99.0 95.3, 95.2, 95.3 100.9,99.5,99.5.98.0 98.4,99.4,96.0, 98.3 101.2, 100.0,99.4 101.5, 100.0, 100.8 98.1, 98.3, 98.2 98.2, 96.6, 98.3, 97.4
99.8 98.6 95.3 99.5 98.0 100.2 100. 8 98.2 97.6
k0.4
10.7 10.1 fl.O 11.3 *o. 8 zO.5 50.3 *0.7
Table IV. Absorbancies at 465 mp, 1-Cm. Cell Substance Calcium pantotheil a t e Niacin
Vitamin A Tocopherol Choline chloride
Serine Threonine Tyrosine Tryptophan Citric acid -4cetic acid Tartaric acid Lactose Glnconic acid Gentisic acid G1u co B e Riboflarin
.
Amount, ME. 0.25 5.0 5.0 5.0 2.5 0.5 0.5 0.5 10.0 2.0 5.0 5.3 5.0 5.0 5.0 5.0 5.0 5 0 5 0 1 0 1 3 2 0 1 0 -a
2-
AUK
Table V.
KO.Detns.
Corn starch, CaPan, Bz Corn starch, CaPan, niacin Corn starch, CaPan, niacinamide Corn starch, niacin, C a P a n Corn starch, CaPan. B?. niacin Corn starch, B I , B2, niacin, CaPan CaPan, Bi, Bzl B6, niacin CaPan, BI, B1. B6, niacin, choline Cornstarch, Bi, Bz, B6,B U Niacin, CaPan, tocopherol, vit. A. CaPan, Bit B2, Be. niacinamide Corn starch, CaPan, B?, Bz, Bs, niacin CaPan, ascorbic acid CaPan,, ascorbic acid, vit. BI, B2, BE,niacin CaPan, ascorbic acid, niacin, tocooherol. vit. A. D. .BI. B2.
4
Table VI.
Sample 0 0 0 0 0 0
Recoveries Obtained on Vitamin Mixtures
Composition
Recovery, % Std. Dev., % 100 1 100 5 101 99 100 0
i l -2 r l &1 &I
4
93 3 99.0 99.0 101.8
a1.3 k2.3 11.4 -1.2
4
99.4
r1.3
4 4
99.1 W.6
11.6 11.0
4 4
100.6 99.4
10.7 r1.7
6 8
6 ,
4 4 4 4 4
8 6 7 2 2
Calcium Pantothenate Found in Vitamin 3Iixtures
Found b y Microbiological .4ssay, Mg./Gram
Found by Chemical Method. Xg,/Gram
Deviation, To
000 010 880 lt5 000 865
0 095
of naphthalenediol reagent of various concentrations. Maximum color was produced with 10 ml. of a 0.1% naphthalenediol solution. Higher concentrations did not increase the color formation. Absorption Curves. Absorption curves of the color developed using solutions of U.S.P. reference standard calcium pantothenate showed a maximum a t 465 mfi measured on a Beckman Model B and Cary recording spectrophotometer. Calcium pantothenate and a,y-dihydroxy &@-dimethyl butyrolactone were first subjected to acid hydrolysis and the hydrolyzed solutions, were used for color development. The absorption curves, Figure 1, show that both calcium pantothenate and the lactone have their maximum absorption a t 465 mp. Stability of Color. Solutions of the colored complex are stable for 18 hours. The method is flexible; after the color has been formed, practically any dilution volume can be used if it is preferable to reduce the sensitivity of the method so that higher concentrations of pantothenate can be determined. Calibration Curve. A plot of the absorbancies against concentration was found to be linear and passed through the origin. Beer’s law is obeyed over a suitable range of concentration, 0 to 500 micrograms. Precision of Method. Samples of calcium pantothenate were assaj-ed by the method described. Very good reproducibility was obtained with a standard deviation of +1.3% (Table 111). PROCEDURE FOR PANTOTHENATE IN VITA3IIN MIXTURES
Experimental. A large number of the substances were tested bj- the procedure described above for pantothenic acid. I n Table IT- are found all the substances tested and the amount of color produced by each. Of the substances most likely to be present i n vitamin preparations, only riboflavin, ascorbic acid, lactose, glucose, and gluconic acid produced colors which interfere in this determination. Riboflavin is removed from the vitamin mixture by chromatographing the aqueous solution of the sample on a Florisil column. The separat.ion obtained is quantitative. In attempts to separate the ascorbic acid from calcium pantothenate. solut.ions of mixtures were chromatographed through
7 8 9 10 11 12 13 14 15 16 17 18 21 22
41 41 4 4 17 19 13 14 17 18 9 8
6 6 7 5 6
42.8 42.1 4.9 4.7 17.9 19.0 13.5 14.0 18.1 18.6 8.6 8.8 32.7 37.0
4
2 1 6 3 1 6 34 1 38 3
Table VII.
+2.8 f1.2 +4.2 -4.4
+t 7
-2. 1 t2.3 -0.7 +2.8
+!.? -a.o
+2.3 -4.1 -3.4
Calcium Pantothenate Assays
Found b y Microbiological Method,
%
Found by Chemical Method,
Sample 23 24 25 26 27 28 29 30 31
101,o 99.8 95.2 99.9 98.0 101.3 101.7 99.7 10‘2.1
99.8 98.6 95.3 99 5 98 0 100 2 100 8 98 2 97 6
70
Deviation, % -1.2 -1.3 4-0.1
-0 4 0 0 -1 1
-
-4
E5
columns of Decalso, talc, Florex, Floridin, Super Filtrol, Amherlite resins IRA400 and IR120, and al minum oxide. Aluminum oxide was found to retain calcium pantothenate which could be eluted by either acid or alkali. However, ascorbic acid is also retained by aluminum oxide and is eluted with the calcium pantot henate. Ascorbic acid, glucose, and lactose are removed by treatment with calcium hydroxide and copper sulfate as described for blood samples (6). The treatment used to remove the ascorbic acid, lactose, and glucose is as follows: The chromatographed solution is transferred to a 125-ml. glass-stoppered Erlenmeyer flask, 1.0 gram of anhydrous copper sulfate is added, and the flask is shaken until all is dissolved. Then 3.0 grams of calcium hydroxide are added and the mixture is shaken and allowed to stand for 30 minutes with occasional shaking. .kt the end of the 30-minute period, the sample is filtered by means of a sintered-glass funnel fitted in a Fisher Filtrator containing a 250-m]. standard taper flask. The original flask and filter are washed with 25 ml. of water. To the comhined filtrate and washings, 5.0 ml. of sulfuric acid are added, the flask is connected to the reflux condenser, and from this point the procedure i 4 eunctly the same as previously descrihed.
1195
V O L U M E 25, NO. 8, A U G U S T 1 9 5 3 In order to be certain that no calcium pantothenate was removed by this treatment, recoveries were seen as follows: h 25-ml. aliquot of standard calcium pantothenate was diluted with 25 ml. of water. Four separate 25-ml. aliquots of the standard calcium pantothenate were diluted with 25-ml. portions of ascorbic acid solutions containing 25, 50, 125, and 250 mg. of ascorbic acid, respectively. Then the five solutions were treated with calcium hydroxide and cupric sulfate as described and the amount of calcium pantothenate in each was determined. Recoveries of 99.6 to 100.270 were obtained. Procedure for Vitamin Mixtures. Weigh an amount of the sample corresponding to about 100 mg. of calcium pantothenate into a 250-ml. glass-stoppered flask. Add exactly 100.0 ml. of distilled water and shake for at least 10 minutes, centrifuge or filter. Place the prepared Florisil column in a Fisher Filtrator containing a 125-ml. glass-stoppered Erlenmeyer flask. Accurately measure a 25.0-ml. aliquot of the clear sample solution onto the column. Collect the eluate in the flask and wash t,he column with successive portions of water, 25 ml. in all. Remove the flask containing the eluate and washings from the filtrator, add 1.0 gram of anhydrous cupric d f a t e , stopper the flask, and shake until all is dissolved. Then add 3.0 grams of calcium hydroxide, shake the mixture. and allow to stand for 30 minutes with occasional shaking. -It the end of the 30-minute period filter the mixture through a sintered-glass funnel fitted in a Fisher Filtrator containing a 250-ml. standard taper flask. Wash the original flask and filter with 25 ml. of water. To the combined filtrate and washings, add 5.0 ml. of concentrated sulfuric acid, connect the flask to o reflux condenser, and gently heat for 1 hour. (Add glass bcads to prevent bumping.) Cool to room temperature and proceed as described for calcium pantothenate. DISCUSSION
The procedures outlined above are based on the requirement of about 250 micrograms of calcium pant'othenate for the development of sufficient intensity of color to minimize instrumental error. By the use of cell paths of greater than 1 em. depth this sensitivity can be inrreased. With suitable modificat,ions based on the nature of the sample. the method should be applicable to many biological materials and food preparations since the separation on the chromatographic column and copper sulfate-calcium
hydroyide treatment have been found to be specific. I t would be necessary, of course, in the use of this procedure on complex biological materials to investigate samples of known concentrations. initially, to preclude the presence of interfering components, CONCLUSIONS
Riboflavin is quantitatively removed from vitamin mixtures by chromatographing on a Florisil column. The copper sulfatecalcium hydroxide treatment completely removes ascorbic acid, lactose, and glucose. The chemical method described was applied to a number of laboratory-prepared mixtures of vitamins and the recoveries obtained are listed. Table V shows the composition of these mixtures with the recoveries obtained. Several commercial vitamin mixtures and feed enrichment mixtures were also tested by the chemical method and compared with the microbiological method. These are shown in Tables VI and VII. Good agreement between the t\vo methods was found. This method is less sensitive than the microbiological method, and, further, the method does not differentiate pantothenic acid and its inactive lactone moiety. LITERATURE CITED (1)
Crokaert, R., and Bigwood, E. T., Arch. intern. physiol., 56,
189
(1948).
Rubin, S. H., AXAL.CHEM, 21, 823 (1949). (3) Feigl, F., Anger, V., and Frehden, O., Miizrochemie, 15, 9 (1934). ( 4 ) Frost, D. V., IND. ENG.CHEX, AN.AL.ED.,15,306 (1943). (5) Saalkowski, C. R., Mader, W. J., and Frediani, H. A , , Cereal 12) DeRitter, E., and
Chcm., 28,218 (1951). (6) Tao, 11.E., Baumann, M.L., and Wask, Shirley, AN.AL.CHEM. 24.722 11952). ( 7 ) Walter, M., Jubilee Val. Dedicated to Emzl Christoph Barell, 1946 98; 2 Vztaminjorach., 18, 228 (1947) (8) Wollish, E. G., and Schmall, M.,A x i L . CHEM.22, 1033 (1950). RECEIVED for review January 3, 1953. Accepted M a y 19, 1933. Presented at the North Jersey Xeeting-in-1Iiniature of the S o r t h Jersey Section of the . h E a I c h r CHEMICAL SOCIETY. S e w a r k , X. 1..January 26, 1053.
Colorimetric Determination of Erythromycin JiRED H. FORD, GEORGE C. PRESCOTT, J. W.IIINJIAIV, AND E. LOCIS CAROU Research Laboratories, T h e Upjohn Co., IGzlamazoo, ItIich.
.4 rapid and precise method for determining erythromycin in all stages of processing from the beer to the crystalline antibiotic was desired in studies on the fermentation and extraction of this antibiotic. The results have been found to be reproducible to about *3% on beers and to about +lyo on aqueous solutions of the purified antibiotic. A chemical assay method which depends upon the development of intense absorption at 485 mp has been found satisfactory for the assay of fermentation liquors and other process samples. Its utility is limited by interference from certain degradation products of erythroniycin, but where the method can be used i t is more rapid and reproducible than the bioassay.
RYTHROLIYCIN is a basic antibiotic having an approximate molecular formula of C31-36H60-6jSOl1-14( 2 ) , which is now in wide use for the treatment of various infections due to Gram-positive bacteria. A rapid and precise method for its determination in all stages of processing from the beer to the crystalline antibiotic was desired to support studies on fermentation and extraction. During some preliminary experiments on degradation, it was obJerved that an intense yellow color was produced when erythromycin was heated with 6 -V hydrochloric acid. The maxiniuni absorption of the solutio~iwa-r found to occur a t about 485
mp. The intact antibiotic has been reported to be tratispnrent in the visible portion of the spectrum and to show only a weak absorption a t 280 mp ( 2 ) . In the authors' first assay experiments, the solutions of erythromycin in 6 N hydrochloric acid were heated for 30 minutes at 50" and the absorbance (optical density) a t 485 nip was measured. However, in subsequent work the hydrochloric acid was replaced by sulfuric acid to reduce the possihility of corroding the spectrophotometer. -2 similar method has been reported for the determination of aureomyin ( I ) , but in this case t'he antibiotic was heated with 0.2 .V hJdrochloric acid and the absorpt,ioii wa3 measured at 440 mp,
