Table I.
Relative Retention Data
Column conditions: 250’ c., r* = 55 p.s.i.g., 20 nil. per minute, 10 inches long
Compound Benzoic acid o-Toluic acid rn-Toluic wid p-l’ciluic acid
:3,.i-l)iiriethylbetizoic acid p-lert-Butylbenzoic acid Phenylacetic acid y-Phenvlbutvric acid 2, ,?-I ) i k ethyi-7-p henylbutyric acid
Kel. ret. time 1 .oo
1.09 1.28 1.31
1.59 2.16 1.32 1.91 3 20
the phenyl alkyl acids chromatographed mere resolved into distinct almost symmetrical peaks. Retention times of all the acids discussed were measured in triplicate under uniform conditions. Table I lists these times relative to benzoic acid. ACKNOWLEDGMENT
The authors thank Sun Oil Co. for permission to publich this work. LITERATURE CITED
(1) Averill, W. J., J . Gas Chromatog. 1,
22 (1963). (2) Burchfield, H. P., Storrs, E. E., “Bio-
chemical Applications of Gas Chromatography,” pp. 527-56, Academic Press, New York, 1962. (3) Byars, B., Jordan, G., J . Gas Chromatog., 2, 304-5 (1964). (4) SlcKinney, R. W., J . Gas Chromatog. 1, 108 (1964). (5) Metcalfe, L. D., Ibid., p. i. (6) Siedermayer, A. O., ANAL.CHEM.3 6 , 938 (1964). JIMT. HILL’ IRAD. HILL Research and Development Sun Oil Co. Marcus Hook, Pa. 1 Present address, Department of Biochemistry, University of Tennessee, hlemphis, Tenn.
Photochemical Changes in Thin Layer Chromatograms of Polycyclic, Aromatic Hydrocarbons SIR: Thin layer chromatography is n o x finding widespread application in t,he separation and identification of polycyclic, aromatic hydrocarbons (1, 4, 8, 12-14> 1 7 ) . Precautions must be taken to avoid photo-decomposition when working with these compounds (6, 9 , 1 1 ) . Also thin layer chromatograms should be developed in the dark, and in the examination of the plates to locate the hydrocarbons, exposure to ultraviolet light should be as brief as possible, to minimize changes in the appearance of t,he fluorescent spots (12) I S ) . Nevertheless, the nature of the changes which may he observed has received litt,le att,ention. We have found that alterations which take place in the appearance of the spots are often the result of photochemical reactions on the thin layer chromatograms. Observations were made on spots of 15 representative hydrocarbons following exposure to ultraviolet light,. The adsorbents used were silica gel G, aluminum oxide G, cellulose powder, and acetylated cellulose (21%). Four of the hydrocarbons (phenanthrene, chrysene, triphenylene, and picene) show no changes after such exposure ot’her than a slight fading of fluorescence when allowed to stand in room light for several days. On silica gel G and aluminum oxide G, pronounced changes occur, both in the appearance and in the fluorescence of the other 11 hydrocarbons. Spots of the hydrocarbons with an anthracenic structure (anthracene, naphthacene, benz[a]anthracene, dibenz [a,c]anthracene, and dibenz [a,h]anthracene) turn yellow or a yellow-tan; while spots of condensed hydrocarbons (pyrene, benzo [alpyrene, benzo [elpgrene, perylene, benzo [ghi] perylene, and coronene) turn darker tan or brown.. At the same time the
appearance of the spots under ultraviolet illumination also changes. The fluorescent color characteristic of each hydrocarbon (usually a shade of blue or green) becomes dull and is gradually lost, while the yiots take on an orange or red coloration, usually changing to such a de&pred that they appear completely dark. h halo (blue, green, orange, or red, depending upon the hydrocarbon involved) is often seen a t the edge of the dark spot. On powdered celluloqe or acetylated cellulose the behavior of these 11 hydrocarbons is somewhat similar, although the changes in appearance and fluorescence are much less extensive and develop much more slowly. After the initial exposure to ultraviolet light, these changes take place even when the plates are kept in the dark. Similar, but slower, changeq also occur on plates kept in ordinary roomlight, without exposure to other ultraviolet illumination. The changes are accelerated by continuous irradiation, either by long-wavelength ultraviolet light or by light of 253.7 mp. The nature of the developing solvent appears to have little effect on the colors observed. However, although it has sometimes been recommended that the chromatoplates be read while they are still wet ( 2 , I S ) , we have found that the presence of solvent often accelerates the changes in the spots. This effect is particularly noticeable with chlorinated solvents. For example, on a plate coated with silica gel G and developed with carbon tetrachloride, a pyrene spot which is examined under ultraviolet light while still wet turns dark and loses much of itq fluorescence within 5 minutes. However, if the plate is allowed to dry for 10 to 15 minutes in the dark before being exposed to ultra-
violet light, the spot is more stable and the blue-green fluorescence is still strong after several hours, although by this time some darkening of the spot can be observed in visible light. I n some instances, this darkening can be utilized in the visualization and identification of hydrocarbon spots. Benzene solutions of pyrene, benzo [a] pyrene, benzo [elpyrene, and benzo[ghilperylene were separately spotted on a silica gel G plate (about 10 p g . of hydrocarbon per spot), developed with heptane, dried in the dark for 15 minutes, and exposed t80 continuous, long-wavelength ultraviolet illumination. The pyrene and benzo [alpyrene spots turned brown within 5 minutes, and all four spots were easily visihle in room light after 15 minutes of ultraviolet irradiation. I n another experiment, the gradual appearance of a brown spot on a silica gel G chromatogram aided in demonstrating the presence of pyrene as an impurity in a sample of chrysene. The reactions which give rise to these changes in the hydrocarbon spots are undoubtedly quite complex. Presumably, such photo-oxidations as have been reported for anthracene on alumina or silica gel (3, 6, 7 , 10, 15, 16) are involved. This is supported by our identification of 1,6-pyrenedione and 1,8-pyrenedione among the numerous products of the reaction of pyrrne on silica gel G. ACKNOWLEDGMENT
The author expresses her appreciation to H. S. Isbell and R . S. Tipson for helpful discussions and counsel, and to A. J. Fatiadi for preparation of 1,6pyrenedione and 1,8-pyrenedione used as reference compounds. VOL. 3 6 , NO. 1 3 , DECEMBER 1 9 6 4
2505
LITERATURE CITED
(1) Badger, G. M., Donnelly, J. K., Spotswood, T. M., J . Chromatog. 10, 397 (1963). ( 2 ) Boyland, E., Sims, P., Biochem. J . 90, 391 (1964). ( 3 ) Craig, D. P., Hobbins, P. C., J . Chem. Soc. 1955, 2309. ( 4 ) Franck-Neumann, M., Jossang, P., J . Chromatog. 14, 280 (1964). ( 5 ) Hoffmann, I)., Wydner, L. C., Cancer 13, 1062 (1960). ( 6 ) Kortum, G., Angew. Chem. 71, 461 (1959). ( 7 ) Kortum, G., Braun, W., Ann. 632, 104 (1960).
(8) Kucharczyk, N., Fohl, J., T'ymgtal, J., J . Chromatog. 11, 55 (1963). ( 9 ) Kuratsune, M., Hirohata, T., S a t l . Cancer Inst. Monograph 9, 117 (1962). (10) Matsumoto, Y., Funakubo, E., 4 i p pon Kagaku Zasshi 72, 731, 733 (1951j; C A 46, 5930d (1952). (11) Moore, G. E., Monkman, J. L., Katz, M., iVatl. Cancer Inst. Monograph 9, 153 (1962). (12) Sawicki, E., Chemist-Analyst 53, 56 (1964). (13) Sawicki, E., Stanley, T. W., Elbert, W. C., Pfaff, J. D., Anal. Chem. 36, 497 (1964). (14) Sawicki, E., Stanley, T. W., Pfaff, J. D., Elbert, W. C., Chemist-Analyst 53, 6 (1964).
(15) T'oyatzakis, E., Jannakoudakis, 11.) Dorfmuller, T., Sipitanos, C., Compt. Rend. 249, 1756 (1959); 250, 112 (1960). (16) Yoyatzakis, E., Jannakoudskis, I).,
Dorfmuller, T., Sipitanos, C., Stalidis,
G., Ibid., 251, 2696 (1960). (17) Wieland, T., Luben, G., Determann, H., Ezperienlia 18, 432 (1962). Pr1.4~S . INSCOE Division of Bureau Of Standards D. 20234 WORKsupported by the Division of Air Pollution, Public Health Service, U. S. Department of Health, Education, and
c.
Welfare.
Chemical Method for Determination of 6-Aminopenicillanic Acid in Fermentation Broths SIR: T o biosynthesize 6-aminopenicillanic acid (6-APA), a chemical intermediate for the preparation of new synthetic penicillins, it is necessary to know the amount of this substance produced for the proper fermentation procesp control. 6-Aminopenicillanic acid has little bioactivity when assayed with penicillin-sensitive test organisms, and therefore direct biological assays are of limited application. An iodometric method for the determination of purified 6-APA has been described ( 1 ) . ,4 determination of 6-APA remaining after extraction of penicillins has been published ( 5 ) . 6-APA has been determined by the difference of the iodometric and biological assays of broths (6). The acylation of 6-APA filtrates and microbiological assay of resulting penicillins has been described (8). h chromatographic method for the identification of 6-ilP4 ( 2 ) and microbiological method for assay of purified 6-APA preparations (3) have been presented. None of these assay procedures is convenient for fermentation process control because they are too timeconsuming or are not applicable to fermentation samples. This investigation presents the method of formation of synthetic penicillins ( 4 ) which can be determined by the hydroxylamine-iron reagent ( 7 ) . EXPERIMENTAL
Reagents and Equipment. All reagents are reagent grade. Amyl acetate-a-phenoxypropionyl chloride is used. AMO;\c-PPC solution is prepared by mixing 0.2 ml. of a-phenoxypropionyl'chloride with 100 ml. of amyl acetate. This is freshly prepared for each use. The reaction vessel is a specially designed screw-capped reactor. It is made of a screw-capped test tube and consists of a reaction chamber, where two phases are mixed, and a short con2506
ANALYTICAL CHEMISTRY
stricted tubing designed for an efficient separation of two phases. The plastic cap permits inversion of the reaction vessel to the position in which the mixing of two phases can be obtained. The total volume of the reaction vessel is about 20 ml. Mixing in a vessel is accomplished with a magnetic bar. Procedure. About 20 ml. of whole broth are transferred to a 22- x 180-mm. test tube and centrifuged a t 1500 r.p.m. for 3 minutes. An aliquot of supernatant solution (10 ml.) is placed in a clean test tube containing a stirring bar and 0.2 ml. of 10% v./v. sulfuric acid. About 10 ml. of chloroform are added and the sample is placed on a magnetic stirrer for 1 minute. -4fter being mixed, t h e mixture is centrifuged a t 1500 r.p.m. for 3 minutes. An aliquot of 5 ml. of aqueous phase (upper) is transferred to the special reaction vessel and 10 ml. of d M 0 . l ~ P P C solution and a Teflon-coated magnetic bar are added. The vessel is covered with a Teflon-lined plastic screw cap. The reactor is inverted (the screw cap is down) and placed on a magnetic stirrer; the contents are
Table I.
Time, min. 0 0 1 2 3 4 5 6 8 10 15 20 25 30 35 40 45 50 60
33 67 00 0 00 00 00 00 00 0
mixed vigorously a t room temperature for 30 minutes. (Six reaction vessels can be placed on the one 8-inch magnetic stirrer.) The reaction vessel is inverted and the plastic cap is removed. Enough time is allowed for the separation of the phases. The bottom acid layer is discarded through a stopcock. Sodium bicarbonate, 2oj, w./v. ( 5 ml.)j is added to the reactor. The reactor is covered with a cap, inverted, and placed on the magnetic stirrer. The contents are mixed vigorously for 30 seconds. The reaction vessel is inverted and uncapped. Enough time is allowed for separation of the phases. The bottom sodium bicarbonate layer (about 3 ml.) is transferred into a vial and is assayed according to the hydroxylamine-iron method ( 7 ) . DISCUSSION
Effect of Temperature. Temperature was found to influence the rate of formation of (wphenoxyethy1)penicillin. .Zn increase in temperature accelerates the rate of reaction, but decomposition of the newly formed
Per Cent Conversion of 6-APA to (a-Phenoxyethy1)penicillin as Function of Temperature and Time
Temp., "C. 4
22.6
25
33.5
47.8
60
70
52 3
63 3 74 3 81 1 84 70.1
70 1 42 1 6 5 85 1 99 100 5
100 5 100 5
67 8 104 97 104 104
1 8 1 1
72 4
59 9 84 4 88 5
92 88 4 104 3
96 1
99 81 3
106 0 96 1
66 87 87 84 45
2 2 8 4
92 1 95 47
100 54 38 5 64 1 59 0 38 5 23 1
