Separation of Aureomycin from Terramycin by Countercurrent Distribution and by Paper Chromatography RICHARD J. HICKEY'
and
WILLIAM F. PHILLIPS
Research and Development Dept., Commercial Solvents Corp., Terre Haute, lnd.
Countercurrent distribution and paper chromatographic techniques have been adapted to analytical resolution of mixtures containing -4ureomycin and Terramycin. The paper chromatographic procedure is primarily qualitative; w-hereas the countercurrent distribution procedure can be utilized on quantitative, qualitative, and preparative bases. The procedures described are very useful tools for accelerating the identification of new- antibiotics similar to Aureomycin or Terramycin in an antibiotic screening program.
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N T H E search for new antibiotics, occasionally one is found which appears to be much like Terramycin or .lureomycin on the basis of early tests on crude materials. The purpose of this study was to determine whether countercurrent distribution and paper Chromatography could be used to separate Aureomycin from Terramycin and whether such methods, if effective, could then be applied to the rapid characterization of unidentified, microbiologically related antibiotics from the screening program. Methods have been described for formation, recovery, and isolation of A\ureomycin ( 5 , 13, 19) and Terramycin (14-16) fIom fermentation liquors. The agents appear to be very similar in certain respects, though there are differences in ultraviolet absorption spectra (8) and in certain functional groups (6, 17). Isolated materials may be differentiated in part by the actio,) of concentrated sulfuric acid (11, 1 8 ) ; a cherry-red color is formed by Terramycin and a blue color by .4ureomycin. The structures of Terramycin (9) and Aureomycin (17, 20) have recently been elucidated. Little information has been published describing useful methods for separating Aureomycin from Terramycin. Countercurrent distribution using a system of n-butyl alcohol with p H 2.5 buffer (14) or with 0.LV hydrochloric acid (16) has been used to purify and characterize Terramycin. Separation of Aureomycin from Terramycin by partitioning in an ethyl acetate-3% aqueous acetic acid system was reported recently ( 7 ) ; however, partition ratios were small. Paper partition chromatography employing Whatman No. 4 filter paper and a solvent system composed of 10% acetic acid, 40% n-butyl alcohol, and 50% water was used in Terramycin studies ( 1 4 ) to show homogeneity of preparations. Sobin et al. ( 1 6 ) also described a method; however, extended zones were obtained for both Aureomycin and Terramycin, and the R, values did not appear t o be critical. Good separation was not reported. Reineke and Peterson (12) have shown that Aureomycin, chloramphenicol, and circulin can be distinguished by chromatography on Khatman S o . 1 filter paper strips with 2y0 p-toluenesulfonic acid in water-saturated n-butyl alcohol as a solvent system. Leach and Teeters (10) separated neamine from neomycin by paper chromatography on Whatman No. 1 filter paper using a solvent system composed of 25y0 glacial acetic acid, 50% n-butyl alcohol and 25% water. Very recently it was reported by Eisenman et al. ( 7 ) that Aureomycin, Terramycin, chloramphenicol, and a new antibiotic, HA-9, could be differentiated using a solvent system composed of n-butyl alcohol, acetic acid, water (4:1:5), but experimental details and completeness of separation were not indicated. 1
The present paper demonstrates clearly that -4ureomycin and Terramycin can be separated readily by both countercurrent distribution and paper chromatographic techniques and that such techniques can be applied to the identification of a prospective new antibiotic suspected of being related. EXPERI % ENTA IL
Countercurrent Distribution Method of Separation. Partitiqning was studied in the glass apparatus ( 4 ) developed by Craig. The lower, aqueous phase, was composed of 0.01,V hvdrochloric acid equilibrated against an equal volume of n-butyl alcohol. The resulting butyl alcohol was employed as the upper phase. Material for distribution consisted of 0.1 gram each of commercial crystalline hydrochlorides of Aureomycin and Terramycin. This was distributed in the aqueous phase of the first four tubes a t the start of the procedure. I n operation, the fundamental procedure described by Craig and Craig ( 4 )was employed, with the volume of each phase being approximately 11 ml. per tube. A total of 100 tubes was used for distribution a t a temperature of 29' C. When the distribution was completed, the content of each tube was shaken with 10 ml. of petroleum ether, and the phases mere separated. The aqueous phases were then analyzed by plate assay ( 3 ) against a sensitive strain of Bactllus rnegatherium using commercial crvstalline Terramycin hydrochloride as the standard. The results are shon-n in Figure 1. Assay values are as Terramycin hydrochloride. Some colored products appeared t o form during the distribution operation. The higher apparent activity of Bureomycin is probably brought about by a grester diffusion rate than for Terramycin, alonr: with t h e use of Terra1.
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TUBE NUMBER Figure 1.
Craig Separation of Aureomycin and Terramycin Hydrochlorides 100 Mg. of each System. n-Butyl alcohol-aqueous 0.01N HCI Number of transfers, 99 Experimental Theoretical
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Present address, University of Pennsylvania, Philadelphia 4, Pa.
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V O L U M E 26, NO. 10, O C T O B E R 1 9 5 4
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mycin as the reference standard for all assays. Sulfuric acid tests and ultraviolet absorption curves of samples of peak material indicated that the activity maximum near tube 52 was Aureomycin, while the maximum near tube 30 was Terramycin. Under the described conditions the calculated partition ratio ( 4 ) for Aureomycin, K,, was 1.11, while that for Terramycin, Kt, was 0.435. Some skewing of the right hand portion of the experimental Aureomycin curve was observed in the comparison with the theoretical curve. The skewed curve may indicate lack of homogeneity of the Aureomycin owing to the presence of an active component closely related to Aureomycin; or it may be caused by a physical phenomenon, not yet determined.
2. Paper Chromatographic Separation. For paper chromatographic separation of Aureomycin from Terramycin the method of Peterson and Reineke ( l a ) was adapted for use with strips of Schleicher and Schuell Yo. 507, ash-free, hardened filter paper. -4satisfactory solvent system was composed of 25% glacial acetic acid, 50% n-butyl alcohol and 25% water (IO).
Table I. Partition Ratios Calculated from Individual Craig Distributions of Aureomycin, Terramycin, and Antibiotic AJ-48 Concentrate NO.
of Transfers 70
.---I j
I
Aureomycin Hydrochloride Room temp., K C. 33-34 1.05
Terramycin Hydrochloride Room temp.,
' C.
K
Antibiotic AJ-48 Concentrate Room temp.,
c.
K
33-34 0,443 33-34 0,522 99 29 1.11a 29 O.43Sa 29 0.524 From distribution of a mixture of Aureomycin and Terramycin hydrochlorides.
1000
E \
u
a J
800
I
-z
E
600
2E w I-
:
400
40
80
120
160
200
TUBE NUMBER
Figure 2. Craig Distribution of a Mixture Containing 120 Mg. of Terramycin Hydrochloride and Antibiotic A 5-48 Concentrate Equivalent to 123 Mg. of Terramycin Hydrochloride System. n-Butyl alcohol-aqueous 0.01.W HCI Number of transfers, 199
The above procedure was applied to a concentrate of an unidentified antibiotic, AJ-48, which appeared to be related to Aureomycin or Terramycin. I n Table I, partition ratios calculated from both 71-tube and 100-tube individual Craig distributions of Aureomycin hydrochloride, Terramycin hydrochloride, and a concentrate of antibiotic AJ-48 are compared. A 5" temperature change had little effect on K values. Sulfuric acid color test and ultraviolet absorption analysis on a sample of the peak fraction from countercurrent distribution of antibiotic AJ-48 failed to distinguish it from Terramycin, but clearly eliminated the possibility of identity ITith Sureomycin. I n order to resolve the question of differences of distribution ratio obtained, a mixture of 120 mg. of Terramycin hydrochloride and a concentrate of antibiotic hJ-48 equivalent in activity to 123 mg. of Terramycin hydrochloride were subjected to a 200-tube countercurrent distribution under the conditions described above. This was considered to be a crucial test. The results, illustrated in Figure 2, show only one activity peak, K = 0.442, indicating the probable identity of antibiotic AJ-48 with Terramycin. The higher partition ratios obtained for AJ-48 concentrates may have resulted from the presence of inactive salts or other impurities.
Along a horizontal line drawn 10 em. from one end of a strip of No. 507 filter paper, 15 X 55 em., were placed 2.5-pI. aliquots of antibiotic solutions by means of capillary pipets. Each aliquot contained from 1.0 t o 1.5 y of Aureomycin hydrochloride, Terramycin hydrochloride, or an equivalent amount of antibiotic AJ-48 concentrate. For resolution of a mixture one aliquot of each antibiotic was applied to the same spot, with drying between applications. After being air-dried, strips were placed in an apparatus for descending paper chromatography consisting of a glass trough supported by a stainless steel frame (Research Equipment Corp., 1135 Third St., Oakland 20, Calif.), enclosed in a borosilicate glass jar 12 inches in diameter by 24 inches in height which was covered with a glass plate. The atmosphere of the jar was saturated with solvent vapor supplied by 200 ml. of the solvent in the bottom of the jar. The strips vere developed for 48 hours a t 25 C. with the acetic acid-n-butyl alcoholwater solvent system, after which they were removed and air-dried. Zones of activity were located microbiologically. The paper stripe were placed on nutrient agar in rectangular borosilicate glass dishes surfaced with a thin layer of agar seeded 15ith a 24-hour culture of Sarcana lutea. After the activity diffused from the strips for 10 minutes a t 25" C., the strips were removed, and the plates were incubated for 18 hours at 37' C. Zones of activity were recorded photographically. The results of a typical comparison of A u r e o m y ci n h y d r o c h 1o r i d e , Terramycin hydrochloride, and a concentrate of antibiotic AJ-48 are illustrated in Figure 3. The Rj values obtained for Aureomycin, Terramycin, SOLVENT FRONT and antibiotic AJ-48 were 0.77, 0.69, and 0.65, respectively. 9mixture of ilureomycin and Terramycin was reFigure 3. Paper solved into t n o distinct oval zones, R, Chromatographic 0.77 and 0.70, respectively. A mixof Separation ture of Terramycin and antibiotic Aureomycin and AJ-48 concentrate gave only one oval Terramycin zone, Rf 0.66, indicating the probable A . 1.5 y antibiotic identity of the t n o antibiotics. Salts AJ-48 concentrate and other impurities in beers and (as Terramycin) crude concentrates apparently affect B . Mixture of 1.5 y of Terramycin hyR/ values considerably; hence, a prodrochloride and spective new antibiotic must be re1.5 y antibiotic solved from mixtures c o n t a i n i n g AJ-48 concentrate C. 1.5 Y Terramycin knox-n antibiotics, before it can be hydrochloride considered as a new substance. D . Mixture o f 1.5 y A4fterthis manuscript had been subTerramycin h y drochloride a n d mitted for publication, the synthesis 1.5 y of Aureoof tetracycline, a new antibiotic rem v c i n hvdrochlolated to Aureomycin and Terramycin . ride E. 1.5 y Aureomycin was reported ( I , 2 ) . Application of hydrochloride the paper chromatographic system Solvent front, 49.1 herein described has resolved tetrac m.
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ANALYTICAL CHEMISTRY ACKNOWLEDGMENT
Table 11. R/ Values of Some Commonly Encountered Antibiotics Obtained by Descending Paper Chromatographya
The authors wish to acknowledge gratefully the technical assistance of J. H. Royer and J. W. Robson.
Antibiotic Rt Streptomycin sulfate 0.06 Streptothricin sulfate 0.06 Netropsin 0.16 Neomycin sulfate 0,006 Arnicetin 0.23 Aureomycin hydrochloride 0.77 Terramvcin hvdrochloride 0.70 ” 5 Schleicher and Schuell No. 507 filter paper and acetic acid-n-butyl alcohol-water (1: 2 :1) solvent system.
LITERATURE CITED
cycline from Aureomycin, Terramycin, and antibiotic .4J-48. Tetracycline showed an Rj value intermediate between those of Aureomycin and Terramycin.
To confirm the preceding evidence of identity with Terramycin, a crystalline hydrochloride with chemical and physical properties identical to those of Terramycin hydrochloride was isolated from an antibiotic AJ-48 concentrate. I n Table 11, for comparison, are listed R , values obtained for purified preparations of some of the more commonly encountered antibiotics using the paper chromatographic technique described above. DISCUSSION
Distinguishing many known antibiotics from each other in crude concentrates is often not difficult because of wide differences in R, values in paper chromatography or because of other characteristic differences. The separation and identification of agents as closely related as Aureomycin and Terramycin, and perhaps others of this class, become somewhat specialized problems, particularly where crude preparations are involved. There are a number of different penicillins, streptomycins, neomycins, and bacitracins. Inasmuch as Terramycin and Aureomycin have been discovered and are so closely related, there is no reason to expect that this class should end with just these two agents. Without adequate methods of separating and identifying members of the group, it is possible that an authentic new agent might be shelved as presumably Aureomycin or Terramycin. ilpplication of the methods described, or modifications, and other criteria ( 4 ) )may be of assistance in simplifying the discovery of new agents of this class.
(16) (17) (18)
(19) (20)
Boothe, J. H., et al., J . Am. Chem. SOC.,75, 4621 (1953). Conover, L. H., et al., Ibid., 75, 4622 (1953). Craig, G. E., private communication, 1952. Craig, L. C., and Craig, D., in -1.Weissberger, “Technique of Organic Chemistry,” 2nd ed., Vol. 3, Chap. IV, New York, Interscience Publishers, Inc., 1950. Duggar, B. AI., U. S.Patent 2,482,055 (1949). Dunitz, J. D., and Robertson, J. N., J . Am. Chem. Soc., 74, 1108 (1952). Eisenman, W., hlinieri, P. P., Abbey, R., Charlebois, J., 3Ioncrieff-Yeates, >I.,and Rigler, N. E., Antibiotics & Chemotherapy, 3, 385 (1953). Hiscox, D. J., J . Am. Pharm. Assoc., Sei. Ed., 40, 237 (1951). Hochstein, F. A, Stephens, C. R., Conover, L. H., Regna, P. P., Pasternack, R.. Brunings, K. J., and Woodward, R. B., J . Am. Chem. SOC.,74, 3708 (1952). Leach, B. E., and Teeters, C. hI., J . A m . Chem. SOC.,73, 2794 (1951). Monastero, F., Means, J. A , , Grenfell, T. C., and Hedger, F. H., J . Am. Pharm. Assoc., Sci. Ed., 40, 241 (1952). Peterson, D. H., and Reineke, L. hf., J . Am. Chem. Soc., 72, 3598 (1950). Pidacks, C., and Starbird, E. E., U. S.Patent 2,586,766 (1952). Regna, P. P., and Solomons, I. A., Ann. N. Y . Acad. Sei., 53, 229 (1950). Regna, P. P., Solomons, I. A., Sfurai, K., Timreck, -4.E., Brunings, K. J., and Lazier, W. A , , J . Am. Chem. SOC.,73, 4211 (1951). Sobin, B. A , , Finlay, -1.C., and Kane, J. H., U. S. Patent 2,516,080 (1950). Stephens, C. R., Conover, L. H., Hochstein, F. A,, Regna. P. P., Pilgrim, F. J., Brunings, K. J., and Woodward, R. B., J . Am. Chem. Soc., 74, 4976 (1952). Van Arkel, C. G., and Weeshoff, A. hI.. Pharm. Weekblad, 86, 389 (1951). Van Dyck, P., and De Somer, P., Antibiotics & Chemotherapy, 3, 184 (1952). Waller, C. W., Hutchings, B. L., Broschard, R. W., Goldman, -1. d.,Stein, W. J., Wolf, C. F., and Williams, J. H., J . Am. Chem. SOC.,74, 4981 (1952).
RECEIVED f o r review February 28, 1953. Accepted June 16, 1954.
Determination of Tin in Titanium Alloys WILLIAM A. DUPRAW Armour Research Foundation, lllinois lnrtitute of Technology, Chicago,
A volumetric method for the direct determination of tin in the range 0.50 to 3.00% in titanium alloys eliminates the acid-sulfide separation of tin from titanium. The sample is dissolved in dilute fluoboric and sulfuric acids. The solution is oxidized with a slight excess of 3 0 q ~hydrogen peroxide. Hydrochloric acid is added and the tin reduced with iron powder. Tin(1I) is titrated with a standard 0.021V solution of potassium iodate.
T
HE best method for the determination of tin is based on
its oxidation from the stannous to the quadrivalent state by means of a standard solution of iodine or iodate. Among the very few interfering substances are nitric acid, vanadium, molybdenum, and tungsten. Vanadium and molybdenum, in their lower valence states, consume oxidant. Lower valence tungsten compounds impart a blue hue to the solution and interfere with
111.
the perception of the starch-iodide end point, but do not consume oxidant. The usual purple color of titanium(II1) would completely mask the starch-iodide end point. However, if a titanium complex could be formed that would be stabIe to the reducers used for converting all tin to the stannous state and yet permit a ready perception of the starch-iodide end point, a direct application of the iodometric method would be possible in the absence of other interfering substances. Other proposed analytical procedures which could apply in whole or in part to the determination of tin in titanium alloys involve either a sulfide separation ( 1 , 4 ) or a distillation procedure whereby tin is volatilized as tin tetrabromide (6). An earlier study was made of the iodometric method and the effect of titanium trichloride (6). An analytical method rn hich permits the direct application of the iodometric method for determining tin in titanium alloys is proposed; this method employs the fluoride complex of titanous titanium. Titanium metal and titanium alloys are easily and
