rose, respectively, from that of the charge. B y changing the ratio of product withdrawal at top and bottom, a n appreciable increase in efficiency of concentration of the sulfur compounds may be realized. Refractive index determinations of the fractions withdrawn from the column at the end of 25 days gave the usual uniform stairstep form of graph typical of a multicomponent miyture, such as a petroleum fraction of wide boiling range. Continuous withdrawal of a top and bottom stream from thermal diffusion columns appears to offer some possibilities for sulfur compound concentration, and study of the problem is continuing. SUMMARY
Liquid thermal diffusion is an effective method for the separation of many classes of sulfur compounds. Continuous withdran-al of sample from the extreme ends of the column is superior to batch operation, in that a greater percentage of a given charge can be separated. ,4n apparatus and technique for charging the column which permits air-free operation are described. A semimicrocolumn (sample capacity, 0.95 ml.) was fabricated and tested. Excellent separations have been obtained with this equipment. Separations by thermal diffusion can
be achieved in many instances on materials m-ith identical boiling points and/or molecular weights without addition of solvents, at the same time avoiding exposure to severe thermal conditions. Thermal diffusion, while not replacing other conventional separation processes, should augment them.
Danby C. J., Lambert, J. D., hlitchell. C. M.. Nature 177, 1225-6- (1956). ’ Grooti S. R. de, “L’Effet Soret,” N. 1‘. Noord-Hollandsche Ultgevers Maatschappij, Amsterdam, 1945. Jones A. L., Petrol. Processing 6, 132-5 (1951). Jones, A. L., Foreman, R. IT., Znd. Enq. Chem. 44, 2249-53
ACKNOWLEDGMENT
Jones, A. L., hlilberger, E. C., Zbid., 45,2689-96 (1953). Jones, R. C . , Furry, IT. H , Rev. AWod. Phys. 18, 151-224 (1946’. Ludnig, C., Sztzber. A k a d . W a s s . Wzen. Math.-nuturw. K1. 20, 539 (1856). Xlelpolder, F. W.,Bronm, R. A . , Washall. T. A , . Dohertv. IT., Young, ’IT. S., ANAL. C H ~ I26, . 1904-8 (1954). Pon-ers, J. E., Ph.D. thesis, University of California, .iugust 1954. Soret, C., Arch. sca. phys. nat., Geneva [3], 2, 48-61 (1879). Stickle, G. P., Ph.D. thesis, University of Tennessee, Sovember 1954. Thompson, C. J., Coleman, H. J . , Rall, H. T., Smith, H. AI,,AVAL. CHEN.27, 175-85 (1955).
\ - - -
(1952).
Grateful acknowledgment is made to Fritz G. RIueller, t o whom this inrestigation is indebted for the design and construction of the continuous sampling units used. RIueller also constructed and contributed to the design of the semimicrocolunin described. ilcknowledgment is also made to Louis Mikkelsen and Don Yee for most of the analytical data and t o Joan Juneau for much of the routine work attending the columns. Finally, the authors wish to thank Harold M. Smith, Regional Director, Region IV, Bureau of Mines (under whose direction this work was originally initiated and partially completed) for his guidance at the beginning and interest throughout the progress of the work.
-
RECEIVEDfor review March 27, 1957. Accepted July 1, 1957. Division 01 Petroleum Chemistry, 131st XIeeting ACS, Miami, Fla., April 1957. Part of the work of American Petroleum Institute Research Project 48h on the production, isolation, and purification of sulfur compounds and measurement of their properties, which the Bureau of Mines conducts a t Bartlesville, Okla., and Laramie, Wyo.
LITERATURE CITED
(1) Begeman, C. R., Cramer, P. L., I n d . Eng. Chem. 47, 202-8 (1955). (2) Clusius, K., Dickel, G., Naturwissenscha..ten 26, 546 (1938).
Differential Spectrophotometric Determination of Novobiocin P I E R 0 SENSI, GIAN GUALBERTO GALLO, and LUlGl CHIESA Research laboratories, lepetif S.p.A., Milano, lfaly A . new method has been devised to detect and to determine novobiocin and dihydronovobiocin individually and in their mixtures. The method i s based on the spectrophotometric examination of the acid hydrolyzates of the two compounds.
N
a new antibiotic, has recently been described. Structure I has been assigned to it (1-10). The hydrogenation product of this antibiotic, dihydronovobiocin, has the same structure except that the double bond in the side chain is reduced. Dihydronovobiocin shows practically the same antibacterial spectrum as novobiocin.
Xovobiocin and dihydronovobiocin have substantially similar chemical and physical properties; melting points, specific rotations, and ultraviolet spectra a t different p H values are identical, while the differences in the infrared spectra are of minor importance. Therefore, it was of interest to devise a
method to determine the two antibiotics in their mixtures. Recently, a paper chromatographic separation of the above antibiotics was recorded (12). When boiled with acids, these antibiotics give two hydrolysis products, novobiocin isoaglycon (cyclonovobiocic acid, 11) and dihydronovobiocin aglycon (111),respectively.
OVOBIOCIN,
I 0
1
H-C-OCONH~
I
AH
\CHZ-CH=C
\
CH3O-C--H
/\
CHS
CHI
/CH3
1
VOL. 29, NO. 11, NOVEMBER 1957
1611
I
I
230
250
300 WAVE
Figure 1. hydroxide
LENGTH
350
370
Sodium dihydronovobiocin, obtained by catalytically hydrogenating novobiocin and purified as for novobiocin. Hydrochloric acid, 6 N . Sodium hydroxide, 0.1N. Ethyl alcohol, reagent grade, 95%. Beckman Model DK recording spectrophotometer with 1-cm. quartz cells. The absorbances can also be evaluated with a Beckman Model DU spectrophotometer. Procedure. About 0.050 gram of the product, exactly weighed, is dissolved in 50 ml. of ethyl alcohol. One milliliter of this solution is diluted to 100 ml. with 0.1.V sodium hydroxide; the absorbance is determined a t 307 mp against 0.1N sodium hydroxide. The percentage of novobiocin (K) and dihydronovobiocin (DN) in the product investigated is
my
Ultraviolet spectra of hydrolyzed antibiotics in 0.1 N sodium
x 100 x 100 6OOX2Xg
A307
xJDN=
------
Novobiocin isoaglycon (cyclonovobiocic acid) Dihydronovobiocin aglycon
The wave lengths chosen are 250 and 330 mp. It is also possible to evaluate a mixture composition by the equations of mixtures (11) from the absorbance values at the above wave lengths.
The ultraviolet spectra of I1 and I11 in 0.W sodium hydroxide are markedly different (Figure 1). The differences in the aglycon spectra appeared sufficient to afford a spectrophotometric characterization of the two products and, hence indirectly, of the two antibiotics. I n fact, by quantitatively converting the antibiotics into the related aglycons, the antibiotics may be characterized either by recording the total spectrum of the two aglycons or by determining the specific absorbances a t suitable wave lengths.
EXPERIMENTAL
Materials and Apparatus. Sodium novobiocin (Vulcamycin Lepetit), purified by several crystallizations from methanol, then dried in vacuo (0.5 mm.) a t 60" C. to constant weight.
where A307 is the absorbance a t 307 mp, 600 is the specific absorbance of both novobiocin and dihydronovobiocin in 0.1N sodium hydroxide a t 307 mp, and g is the amount of the product in grams. Then 25 ml. of the ethyl alcohol solution is transferred to a 100-ml. flask, 10 ml. of 6 N hydrochloric acid is added (the acid concentration in the mixture is about 1.71N), and the solution is refluxed for 2 hours on a water bath; 50 ml. of ethyl alcohol is then added, and the solution is cooled and diluted to 100 ml. with alcohol. One milliliter of this solution is diluted to 25 ml. with 0.1N sodium hydroxide and the absorbance values a t 250 and 330 mp are determined against 0.LV sodium hydroxide in 1-cm. quartz cells. Calculations. The following values were obtained for the specific absorbances in 0.1N sodium hydroxide of novobiocin and dihydronovobiocin solutions after acid hydrolysis; they are the average of 18 experimental determinations. Kave Length, M p
E%.
250 330
524 440
Novobiocin Std. dev. 5.1 5.0
Dihydronovobiocin Std. dev. E;%. 250 330
336 550
4.4 5.7
The ratios between the specific absorbances a t 250 and 330 mp are CJ33
I11
Xovobiocin
= 524 -=
Dihydronovobiocin
16 12
ANALYTICAL CHEMISTRY
1.191
440 336
= 550 - = 0.611
Table 1.
Analysis of Novobiocin-Dihydronovobiocin Mixtures
Weight of Sample, Grams DihydroPu'ovobiocin novobiocin 0.04997 0.04704 0.04090 0.02294 0.02245 0.00613 0.00156
0.00494 0.01234 0.02392 0.03704 0.04069 0.04793 0.05132
...
+Novobiocin Dihydronovobiocin Found,
% 99.7 99.3 100.4 90.2 99.4 100.5 99.7 99.8
Novobiocin, % Dihydronovobiocin, 70 Calcd. Found Calcd. Found 100 90.5 76.8 48.9 37.8 13.1 3.2 0.0
From the ratio of the two readings at 250 and 330 mfi, it may be ascertained whether the product investigated is novobiocin, dihydronovobiocin, or a mixture of both. The antibiotic concentration in the mixture is calculated as follows.
+ Cz 336 C1440 + Cz 550
99.3 90.4 73.7 50.0 37.2 13.2 3.2 1.1
0.0 9.5 23.2 51.1 62.2 86.9 96.8 100.0
yo dihydronovobiocin
1.2 9.1 25.0 49.5 64.1 86.1 97.6 100.2
=
(0.0037332 X A m - 0.0031348 X A250) x 100 x 100 2 x g
where g is the weight of the sample in grams.
A250 = CI 524 -4330
RESULTS AND DlSCUSSlON
where AZmand A330are the absorbances of the final solution against 0.1N sodium hydroxide at 250 and 330 mp; C1 and Cz are the concentrations (in grams per 100 ml.) of novobiocin and dihydronovobiocin, respectively, in the final solution; and 524, 440, 336, and 550 are the specific absorbances. B y solving these two equations, the following values are obtained: C1
0.0039185 X A250
0.0023938 X Asaa
Cz
t
0.0037332 X A330
0.0031348 X
8250
The percentages of novobiocin and dihydronovobiocin in the sample are
9Gnovobiocin
=
(0.0039185 X A m 0.0023938X Asso) X 100 X 100 2 x g
Mixtures of novobiocin and dihydronovobiocin prepared from standard samples were evaluated; the results obtained are summarized in Table I. The conditions were established after the following factors affecting the reaction were investigated. Acid Concentration. With 3 to 4Ahydrochloric acid a high conversion rate was obtained, b u t partial degradation of t h e hydrolysis products was noted. When 1.71N acid was used t h e hydrolysis was complete in 90 t o 120 minutes. Results were constant, with no side reactions. Lower acid concentrations prolonged t h e reaction time too much for practical purposes. Temperature. B y carrying out t h e reaction at 40' C., t h e two hydrolysis products from novobiocin and dihydronovobiocin showed identical ultraviolet spectra. At this temperature novobiocin aglycon iprobably
does not undergo t h e ring closure to cyclonovobiocic acid. It was convenient t o carry out t h e reaction at t h e boiling temperature of t h e hydrochloric acid-ethyl alcohol mixture. Alcohol-Aqueous Hydrochloric Acid Ratio. Novobiocin, dihydronovobiocin, a n d their acid hydrolyzates a r e insoluble i n aqueous hydrochloric acid. T h e addition of ethyl alcohol is necessary, therefore, t o solubilize t h e products. On other hand, t h e course of hydrolysis in ethanolic acid media is abnormal. For a 25-mg. sample, a mixture of 25 ml. of alcohol and 10 ml. of 6N hydrochloric acid is suitable. Reaction Time. T h e hydrolysis was followed under t h e conditions selected b y removing a n aliquot of t h e boiling mixture from time t o time, diluting to a suitable concentration, and determining t h e absorbance in 0.1N sodium hydroxide. The absorbance values were constant from 1.5 to 3 hours (Figure 2). Therefore, the reading was made after 2 hours' heating. The solutions, in concentrations from 2 to 12 y per ml., follow the LambertBeer laiv. LITERATURE CITED
(1) Hinman,
J. W., Hoeksema, H., Caron, E. L., Jackson, W. G.,
J . Am. Chem. SOC. 78, 1072
flS.i6). (2) Hoeksema, H., Bergy, M. E., Jack\ - - - - ,
son, W.G., Shell, J. R., Hinman, J . W.,Fonken, A. E., Boyack, G. -4..Caron. E. L.. Ford. J. H.. DeVries, W.' H., drum, 'G. F.; Antibiotics & Chemotherapy 6, 143 (1956).
(3) Hoeksema, H., Caron, E. L., Hinman, J. W., J . Am. Chem. SOC. 78, 2019 (1956). (4) Hoeksema,
H., Johnson, J. L., Hinman, J. W.,Ibid., 77, 6710
(1955). (5) Kaczka, E. A , , Shunk, C. H., Richter, J. W., Wolf, F. J., Gasser, M. M., Folkers, K., Zbid., 78, 4125 (1956). (6) Kacaka, E. .4.,Wolf, F. J., Rathe, F. P.. Folkers., K.., Zbid.., 77.. 6404 (1955). (7) Rolland, G., Senei, P., De Ferrari, \ - - - - I
G. A., Maffii, G., Timbal, M. T., Silvestri, L. G., Farmaco 11, 549
(1956). (8) Shunk, C. H., Stammer, C. H.,
