0.399 for transition times of 0.026 to 303 seconds, whereas the corrected values show a variation of only 0.391 to 0.398 over this 12,000-fold transition time range. It is difficult to determine what fraction of B is made up of the various factors it contains. I n all of the cases examined the contribution from charging of the double layer was probably within 0.01 millicoulomb. Oxidation of the electrode was very important during the electro-oxidation of hydroquinone. Oxidation in a pH 7 buffer alone shows that a platinum electrode starts to oxidize in the potential range where h:, droquinone oxidation occurs, and contributes a t least 0.1 millicoulomb to B. The additional contributions to B for the other substances may reflect adsorption of the electroactive species of about 2 X moles per sq. cm. (about one tenth of a monolayer), although only small amounts of contribution from oxide film effects would make this estimation seriouslv in error. Laitinen has recently suggested (5)
that chronopotentiometric measurements may be useful in determining the amount of adsorbed electroactive species on an electrode. The method of treating the data which he suggests, plotting i0r us. l/&, which implicitly assumes that the electrolysis of the adsorbed species occurs first, can also be used in correcting experimental data. Plotting our experimental data according to this scheme vielded straight lines with a slope giving essentially the same value of the chronopotentiometric constant, but with resulting B values about twice as large. The obvious difficulty with this type of measurement, as illustrated by the results given here, is that it is difficult to determine what contribution is made by oxide film effects to B. Chronopotentiometric measurement of adsorption by this technique would probably be more useful on mercury electrodes, and the most fruitful approach on solid electrodes is probably that taken by Lorenz and coworkers (7, 8), which involves heavy platinization of the electrode, increasing
the adsorption effect, and yielding variaof up to 800%. tions of i,rl/z/CO ACKNOWLEDGMENT
The author gratefully acknowledges the support of the Robert A. Welch Foundation. LITERATURE CITED (1) Anson, F. C., ANAL.CHEM.33, 1123 .
I
(1961). (2) Anson, F. C., Lingane, J. J., J. Am. Chem. SOC.79, 1015 (1957). (3) Bard, A. J., ANAL. CHEM. 33, 11 (1961). (4) Delahay, P., “New Instrumental Methods in Electrochemistry,” p. 207, Interscience, New York, 1954. (5) Laitinen, H. A., ANAL.CHEM.33, 1463 (1961). (6) Laitinen, H. A., Enke, C. G., J. Electrochem. SOC.107, 773 (1960). (7) Lorenz, W., 2. Elektrochem. 59, 730 119.55\. (8) Lolrenz, W., Muhlberg, H., Ibid., p. \ - - - - / -
7.2fi
(gjviikinmuth, W. H., ANAL. CHEM.33, 323 (1961). RECEIVED for review September 7, 1962. Accepted December 21, 1962.
Thin Layer Chromatography of the Mixed Neomycin Sulfates on Carbon Plates T. F. BRODASKY Research laborafories, The Upjohn Company, Kalamazoo,
b To facilitate the analysis of commercial preparations of neomycin sulfate, a novel method for the chromatography of neomycins A, B, and C sulfates has been developed. The procedure is based on the application of thin layer chromatography on carbon black, using aqueous acid as the mobile phase. The separation has been carried out on carbon pretreated with HzS04 and on untreated carbon. Both substrates are effective when using an acidic mobile phase; however, water as the mobile phase fails to resolve the B, C sulfate complex. The effects of treatment of the carbon and varying the nature of the mobile phase are discussed in relation to their influence on the separation of the antibiotics. The biological activity on the thin layer plates was detected using the agar diffusion technique with Bacillus pumilus, and the quantitation of the neomycins on the organism is described. The quantitation of the neomycin sulfates proved to be difficult because of stimulation of the organism due to certain experimental techniques. This problem was partially resolved by adjustment of the inoculum rate and incubation temperature.
S
Mich.
of neomycin by Waksman in 1949, a direct technique for the analytical separation of two stereoisomeric forms, neomycin B and neomycin C, has not been available. For some time, workers separated neomycin B from its degradation product, neomycin A (neamine), by paper chromatography without effecting a separation of the stereoisomers (5, 6) although there was evidence for the presence of these compounds in the neomycin complex (8, IO). I n 1951, Dutcher et al. (g) separated the hydrochloride salts of neomycin B and neomycin C on an alumina column, eluting with 80% methanol. Ford et al. (3) were successful in separating the two isomeric sulfates on an acid-treated carbon column, using water as the eluent. Although the antibiotics had been separated on preparative adsorption columns, an analytical procedure using paper chromatography was not readily developed. Saito and Schaffner reported the separation of neomycins A, B, and C on paper saturated with 1.OM sodium sulfate, using aqueous methanol containing sodium chloride as a developing solvent (9). However, the method proved less than satisfactory. The conversion of the antibiotics to their N INCE THE DISCOVERY
acetyl derivatives did result in a satisfactory separation of the three compounds and to date has been the only analytical procedure for the separation of the neomycins ( 7 ) . This paper introduces a novel qualitative method of chromatographing the neomycin sulfates based on the application of thin layer chromatography on carbon black. The quantitative aspects of this technique have presented considerable difficulty. Measures designed to overcome some of these problems are described. EXPERIMENTAL
Preparation of Carbon. Thirty grams of Nuchar (C-190-N) vegetable carbon black and 1.5 grams of CaSO4. 1/2 HzO were slurried with 220 ml. of distilled water or distilled water adjusted t o p H 2 with HzS04. The carbon prepared with distilled water was applied to the glass plates immediately; however, the acidified carbon must stand a minimum of 16 hours prior t o preparation of the plates. Using these quantities i t is possible to coat 35 plates. Preparation of Thin Layer Plates. Single-strength glass plates (100- by 200-mm.) were degreased by washing with acetone. The carbon was applied with a Camag thin layer apVOL 35, NO. 3, MARCH 1963
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RESULTS AND DISCUSSION
Figures 1and 2 show the separation of neomycins A, B, and C sulfates on untreated and acid-treated carbon thin layer plates developed in 0.5N H2SOa. The relative position of A with respect to the parent compounds is m accord with the empirical rule that as the C to N ratio decreases the R, on carbon increases. The C to N value for neomycin A is 3.0 and for B and C is 3.8. Table I gives the R, values encountered when chromatographing neomycins A, B, and C sulfates under the various conditions used in this study. The specificity of the separation on carbon was demonstrated when thin layer chromatography using silica gel and cellulose failed to resolve neomycins B and C and gavc only partial resolution of A from the B, C complex. Figure 1 .
Chromalograms
of neomycins on untreated carbon Table I. R, Valuer of the Neomycinr with Various Chromatographic Systems
plicator (Arthur H. Thomas Co.) using a film thickness of 3 X 10-2 cm. The plates were air-dried and, without further treatment, stored on plate racks in stainless steel containers. Chromatography. For qualitative studies the antibiotic sulfates (from this point on in the text, the terms neomycin A, B, or C specifically refer to the sulfate salts and not the free bases) were normally applied a t levels of 5 pg. of neomycin B, 10 pg. of neomycin C, and of 15 pg. of neomycin A. For quantitative experiments neomycin B standard was applied a t 2 , 4 , 8, and 10 @g.with samples at 60 to 70 pg. This was done because neomycin B samples were generally being assayed for neomycin C which was present at levels of 257, or less. Neomycin C was applied at 7, 10, 15, and 20 pg. while neomycin A standards were applied at 10,15,20, and 25 gg. to demonstrate the standard curve. The standard curves were constructed by plotting the log dose os. zone diameters on a detecting organism. Plates were developed in either water or 0.5N acid to within 50 mm. of the top of the plates. After drying they were neutralized by exposure to a n ammonia atmosphere for a minimum of 5 minutes. This prevented inhibition of the organism by residual acid. The biological activity on the plates was detected using the agar diffusion method with R. pumilus. Trays (19- X 50-em.) containing 125 ml. of streptomycin agar seeded with 2 X lo5 cells per ml. were used, each tray holding four plates. The plates were applied and pressed firmly onto the agar surface. After 5 to 10 minutes thev were removed a.nd t,hr
of the agar.. It is important not t o attempt its removal until after incubation, as antibiotic material may be -spread to other2portions of the tray.
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ANALYTICAL CHEMISTRY
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'
After incubation, the surface of the agar was wetted and a microscope slide was gently drawn across the area where carbon was retained. This resulted in a clean, undamaged surface. The agar diffusion method from thin layer plates employed routinely in this laboratory has been successfully applied to antibiotics in general. Parallel experiments with papergrams have shown no evidence of excessive adsorption of antibiotics on thin layer matrices including such materials as cellulose and silica gel. No adverse effect on the detecting organisms has been demonstrated, with the exception of tissue culture which does not tolerate silica gel. This method provides a necessary means of specifically detecting biological substances on thin layer plates.
Figure 2.
Acidified carbon Untreated carbon developed with developed with Neo0 5N 0 5N mycin HzO HsSOI H,O H&O4 A 0.60 0.61 0.00 0.54 B 0.10 0.21 0.00 0.24 '
C
0.10
0.43
0.00
0.45
Consideration of these data led to some important conclusions concerning the separation. Neomycins B and C were completely resolved in the 0.5N HSO, system on Untreated and aoidtreated carbon whereas acid-treated carbon developed in water was ineffective in the separation of the antibiotics. This indicates that the separation is dependent on the presence of acid in the
Chromatograms of neomycins on acid-treated carbon
20
,,
-
i
t
9’
/ /
/
/
Y
_ _ _ _ _NEOMYCIN -
A
NEOMYCIN B NEOMYCIN C
I 2
0
/ 4
12
8
16
20
24
ZONE D I A M E T E R (rnrn.)
Figure 3.
Standard curves for the neomycins on B. pumilus
mobile phase of the chromatographic system. If this is true, the separation should also be accomplished in the presence of mineral acids other than H2S04. h’eomycins B and C chromatographed on carbon pretreated with p H 2 HC1 using 0.5N HC1 as the mobile phase gave Rf values duplicating those obtained with sulfuric acid. Under such conditions the amine and hydroxyl (electronrich) groups would be protonated and excess acid in the mobile phase would deactivate adsorption sites on the carbon, thus reducing antibiotic adsorption. Chromatography could then proceed governed principally by the stereochemical differences in the molecules. Acid-treated carbon developed in water does not fulfill the required condition of having acid in the mobile phase. -4developed plate of acid-treated carbon not containing antibiotic and which was not neutralized showed no inhibition on B. pumilus (untreated carbon developed in 0.5N H2S04showed strong inhibition under these conditions) indicating the absence of free acid. The acid present on the plate is apparently not available to the mobile phase because of adsorption. If one aswmes a specific surface of 106 sq. cm. per gram ( 1 ) for the carbon black, calculations slion that on each plate (containing 0.7 gram of carbon) only 10% of the carbon surface is covered by acid molecules. The evidence indicates that this is not sufficient acid to cover the active sites and acidify the mobile phare. Plating of untreated carbon plates developed in acid but not neutralized resulted in inhibition of B. pumilus from the lower edge of the d a t e to a n R,
of 0.5 to 0.7. The adsorption of acid to the saturation point was slow enough to allow the acid-depleted water to move ahead of the acid front. Under normal conditions of development, neomycin A travels near the acid front. Therefore, when working with substances in this region, it is best toallow?the solvent front to reach the end of the plate to effect a greater separation between neomycin A and the acid front. The alternative is to develop the antibiotics on acid-treated carbon. Although the neomycins are separated satisfactorily on untreated carbon with 0.5N acid, it is advisable when using less than 10 pg. of neomycin A or neomycin C to effect separations on acid-treated carbon, as larger zones are obtained. Quantitation. Figure 3 shows the standard curves obtained with neomycins -4,B, and C on B. pumilus seeded agar. I n general, little difficulty was encountered i n the quantitation of neomycin B since B. pumilus was sensitive t o this component down to 2 ug. Xeomyein A, on the other hand, required about 10 times the dose rate of neomycin B for suitable zone sizes. Keomycin C presented some problems resulting from the marked variation in response of the organism. Initial experiments were carried out using Bacillus subtilis until it was determined that the response of both neomycins B and C was increased on B. pumilus. Although the response was greater for both antibiotics, neomycin B was still by far the more active and in large applications interference of the B, C zones was noted. The greatest difficulty in detecting low levels of neomycin C was attributed t o
the pronounced stimulation of the B. pumilus organism due to a combination of the carbon substrate and formation of ammonium ions after neutralizing the plates. Attempts to overcome this effect led to the u5e of the present level of B. pumilus inoculum. Incubation at 28 a C. also helped to reduce the stimulation effects. Consideration of the experimental procedures and the data from various assays leaves the accuracy of this technique in question. The nonuniform zone shapes and, in some cases, anomalous responses, especially from neomycin C and A requires a good deal of experience on the part of the operator t o obtain reproducible results. It is advisable to report results based on total antibiotic present as determined by individual assays rather than the total weight of material used. Results obtained in this manner and by using more accurate methods are in better agreement than those based on total weight of samples. Although the carbon thin layer quantitation is of questionable accuracy, the separation of the neomycin antibiotics and the quantitation should be a powerful adjunct to the highlyaccurateradioactive tracer quantitations now being used in this laboratory ( 4 ) . ACKNOWLEDGMENT
The author expresses his gratitude t o
R. W. Rinehart and G. B. Whitfield for their many helpful discussions and t o Joyce Leniin and Patricia Barton for their untiring efforts in preparing, spotting, and developing the carbon thin layer plates. LITERATURE CITED
(1) Bickerman, J. J., “Surface Chemistry,” 2nd Ed., P-220, Academic Press, 1958.
(2) Dutcher, J. D., Hosansky, N., Donin, M., Wintersteiner, O., J . Am. Chem. SOC.73,1384 (1951). (3) Ford, J. H., Bergy, M. E., Brooks,
A. A., Garrett, E. R., Alberti, J., Dyer, J. R., Carter, H. E., Zbid., 77, 5311 (1955). (4) Kaiser, D. G., ANAL. CHEM. 35, in Dress. (5) Leach, B. E., DeT’ries, W.H., Nelson, H. A., Jackson, W. G., Evans, J. S., J . Am. Chem. Sac. 73, 2797 (1951). (6) Leach, B. E., Teeters. C. M., Ibid., 73, 2784 (1951). ( 7 ) Pan, S. C., Dutcher, J. D., ANAL. CHEM.28, 836 (1956). (8) Regna, P. P., Murphy, F. X., J . Am. Chem. Sac. 72, 1045 (1950). (9) Saito, A., Schaffner, C. P., Angew. Chem. 67, 666 (1955). (IO) Swart, E. A., Hutchison, D., Waksman, S. A,, Arch. Biochem. 24, 72 (1949). RECEIVED for review September 24, 1962. Accepted December 20, 1962.
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