A N A L Y T I C A L CHEMISTRY
744 where t is temperature in degrees Centigrade. This wag found to be accurate within the experimental limits of the method. INTERFERENCES
Table 111. Determination of Iron and Vanadium in Titanium Tetrachloride
2
3
Source 2 nil. 0 0513.21 FeCla 10 ml. Cenro tech. TiCL 4 ml. 10 ml. 1 ml. 10 ml.
Iron, M g . Calcd. Found 5 73 5.7
0 0196.W FeCla Cenco tech. TiClr
4.38
0.0196.WFeCls Cenco tech. TiClr
4 38
I
5 00
The only interference studied was that of iron, as it was considered the most probable impurity besidee vanadium. S o iron was found in the commercial samples of titanium tetrachloride, EO synthetic samples were prepared by adding a standard solution of ferric chloride to a hydrolysed solution of Cenco technical titanium tetrachloride. Acid strengths were the Pame as those used for the determination of vanadium alone.
Run 1
.-I‘
-1.3 4.3
_-
Vanadium. lfg, Calcd. Found 12.62 12.6 12.62 12.62
12.8 12.8
Figure 3, A , shoJvs the titration curve a t a wave length of 190 mp, and Figure 3, B, s h a m the titration curve a t 760 mp. T h e presence of iron causes a flattening of the vanadium(1V)vanadium(II1) absorbance line near the second end point, thereby making the end point ~ O I difficult P to determine. By using t h e straight portion of the lines near the vanadium( 1V)-vanadium (111) and iron(II1)-iron(I1) end points, fairly accurate results for both elements can be obtained although they are not so accurate as for vanadium alone. .1 possible improvement muld be made by adding a reagent which would form a ferrous compIex of high absorbance. Table I11 shows the results for analyses of per cent vanadium and iron in the same titanium tetrachloride solution. Titrations 1 and 2 were made a t 490 my and titration 3 was made a t 760 my. The iron solutions wei e standardized n-ith potassium dichromate.
em
m IO.
51
703
800
KO
400
333
ar,
loo
3 INCHES
205-
-
0
-(Fet:Fet2)
_ -~
-
(V ‘4V t3 ) - __..-
-(Vf4V+4) -_ -_
ACKNOWLEDGMENT
The authors are indebted to Cramet, Inc., Chattanooga, Tenn., for financial support of this project. LITERATURE CITED
(1) Arthur, P., and Donahue, J. F., ANAL.CHEM.,24, 1612 (1952). (2) Goddu, R. F., and Hume, D. N., I b i d . , 22, 1314 (1950). (3) Kolthoff, I. M.,and Furman, N. H., “Potentiometric Titrations,” 2nd ed., p. 373, Wiley, New York, 1931. {4) Malmstadt, H. V., and Gohrbandt, E. C., ANAL.CHEM.,26, 442 (1954). (5) Reilley, C. N., Cooke, W. D., and Furman, N. H., Ibid., 23, 1030 (1951). (6) Reilley, C. N., and Schweizer, B., Ihid., 26, 1124 (1954). (7) Sabatini, R., Hazel, J., and McNabb, W., A n a l . Chim. A c t a , 6, 368 (1952). (8) Sagawa, T., Complete Abstr. J a p a n . Chem. Lit.,6, 341 (1932). (9) Shirrer, G., Johnston, H. L., and Beckett, C., “Titanium and Its Compounds,” p. 43, Herrick L. Johnston Enterprises, Columbus, Ohio, 1954. (10) Sweetser, P. B., and Bricker, C. E., ANAL. CHEM.,24, 409 (1952). (11) Ihid.. 25. 263 (1963). tl2j Underwdod, A: L., Ibid., 25, 1910 (1953). (13) I h i d , , 26, 1322 (1954). (14) Wise, E. N., Gilles, P. TI7., and Reynolds, C. A., Jr., Ibid., 25, 1344 (1953). RECEIVED for review September 20. 1954. Accepted December 30, 1QZ-1.
Determination of Erythromycin by Ultraviolet Spectrophotometry JOHN
B. TEPE
and
C. V. ST. JOHN
Eli Lilly & Co., Indianapolis, lnd. I
The development of an accurate method for the determination of erythromycin w-as based on the observation that alkaline hydrolysis produced a material that had a characteristic ultraviolet absorption. An acid-inactivated blank was employed to correct for the ultraviolet absorbing impurities and degradation products of erythromycin. The assay has shown good reproducibility, sensitivity, and correlation with a turbidimetric microbiological assay.
E
RYTHROMYCIN ( 7 )is a basic antibiotic that is used estensively for the treatment of various bacterial infections. An accurate method of assay was desired for processing and formulation, during the manufacturing of this antibiotic. A colorimetric assay for erythromycin reported b y Ford and coworkers ( 8 ) employed sulfuric acid to develop a color that absorbed at 485 mp. Acid-hydrolyzed erythromycin (8) reacted with NelEon’s reagent to develop a blue color. These methods do not appear to be specific for erythromycin.
During the course of t,his investigation for a chemical method of analysis, many color reactions were studied. Those that gave positive reactions with pure erythromycin are listed with comments as to their usefulness in analyzing samples of varying purities that are encountered in the fermentation and purification of the antibiotic. The procedures and conditions employed were the same as described in the literature. 1. Erythromycin when heated with a mixt,ure of glacial acetic acid and perchloric acid (varying proportions) gave a color that absorbed strongly at 485 my. Reproducibility was good but the test gave higher values on process samples than microbiological assay or the ultraviolet assay. 2. Erythromycin mas oxidized by periodic arid and the resultant, formaldehyde vas determined by chromotropic acid ( 2 ) . The reproducibility was good but the test was not sufficiently sensitive for dilute solutions. 3. The tryptophan-perchloric acid reagent. ( 3 ) reacted with erythromycin t o produce a red color. This method had good sensitivity and reproducibility; however, degradation products of erythromycin in impure samples reacted with the reagent. 4. The xanthydrol reagent ( 9 ) reacted with the erythromycin to give a red color that absorbed strongly at 540 my. The sensi-
V O L U M E 27, NO. 5, M A Y 1 9 5 5
745 Diglycolic avid (Matheson, Colemaii and Bell, 5973), 0.1% aqueous solution, with pH adjusted to 4 0 with 1-V sodium hydroxide, RECOMMENDED PROCEDIIRF
WAVE
LENGTU
my
Figure 1. Absorption spectra of erythromycin 1.
2.
3.
Aqueous solution, 1000 y per nil. Five-milliliter sample containing 1000 y, hydrolyzed with 1.0 nil. of 0,05>V NsOH. a t 100' C.. 5 minutes, final dilution 100 y per nil. Acidinactivated blank, 100 y per nil.
tivity and reproducibility were good; however, degradation products from erythromycin caused variable results. Other reactions were investigated and found useful for the analysis of pure samples but. were not applicable for process samples because of a lack of sensitivity or specificity. The reactions st'udied included the treatment of erythromycin with p-h?-droxybiphenyl ( 1 )> Folin-Ciocalteau phenol reagent ( 5 ) , cliphenylamine in acetic acid and sulfuric acid ( 4 ) ,phosphomolybdic acid ( I t ) ? and diazotized sulfanilic acid and sodium hydroxitle (11). .liter considering the above procedures, i t was found that. the characteristic ultraviolet absorption properties of hydrolyzed er>.thromycin offered the most promising method for analysis. I:r>,thromycin exhibits a broad weak absorpt,ion band (Figure 1 i i n the ultraviolet a t 285 nip. After strong acid hydrolysis at elevated temperatures (Figure 2 ) . erythromycin exhibit's maxima :rt 226. 267, and 485 nip. The absorption at. 226 m,u has an of approximately 150 and obeys Beer's law. Other degr:i(littion products of erythromycin also have absorption in the ?%-nip range and limit the usefulness of acid hydrolysis as an a
