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Effect of Temperature on the Separation of Conformational Isomers of Cyclic Nitrosamines by Thin-Layer Chromatography Haleem J. Issaq,' Mario M. Mangino, George M. Singer, David J. Wilbur, and Nelson H. Risser Chemical Carcinogenesis Program, Frederick Cancer Research Center, Frederick, Maryland 2 170 1
The separation of conformational isomers of cyclic nitrosamines which did not separate at room temperature was possible when the thin-layer chromatography (TLC) plate was developed at -77 OC. Effects of temperature and continuous development on the separation and resolution were studied. The results show better separation at lower temperatures and improved resolution with continuous development. NMR spectra of the separated compounds support the TLC data which indicated that the separated compounds are conformational isomers.
Separations by thin-layer chromatography (TLC) are normally carried out a t room temperature. However, in certain cases it may be necessary to develop the plate a t lower or higher temperatures than room temperature in order to achieve a separation. I t has been shown that lower temperatures affect the separation of compounds by TLC. Abbott e t al. (1) studied the effect of temperature (+40 "C to -20 "C) on development times and R, values of 12 chlorinated pesticides. They found that the migration rates of all compounds studied were temperature dependent. Compounds with R, values above 0.40 at room temperature showed the greatest variation; compounds with R, values of less than 0.20 showed almost no variation at higher temperatures although their R, values dropped a t -20 "C. Separation was least effective a t -20 "C. Abbott et al. (1)and Stahl(2) observed that development of the plates at low temperatures was not only faster but also that more compact spots were obtained, which resulted in better resolution. Stahl ( 3 ) was able to separate a mixture of geranyl acetate, asarone, carotol, and hydroxycaryophyllene at -10 "C, which is otherwise difficult. Henderson and Clayton ( 4 ) used ultra cold chromatography to separate saturated phosphoglycerides. Lieberek e t al. (5) used TLC a t 0-2 'C to separate E and Z isomers of N-nitroso-N-alkyl-amino acids. They also reported that TLC a t 0-2 "C offers a more sensitive method of checking the conformational purity of isomers than does NMR spectroscopy, since small contamination of one by the other can be detected more easily by TLC than by NMR spectroscopy. In our work it was necessary to develop a method for the separation of conformational isomers of heterocyclic nitrosamines which are in equilibrium a t room temperature owing to rotation about the N-N bond of the nitrosamine function. I t seemed likely that if the rate of rotation could be slowed by reducing the temperature, then the difference in polarities of the conformers might allow separation by TLC. This paper describes a cryogenic apparatus used for the separation of conformational isomers a t -77 "C. NMR and TLC data show that the separated compounds are conformational isomers. T o our knowledge, this is the first instance of a separation of nitrosamine conformers in which the equilibrium is free from the influences of hydrogen bonding ( 5 , 6 )or steric effects (7). 0003-2700/79/0351-2157$01.00/0
EXPERIMENTAL Apparatus. Standard glass tanks were used for plate development at room temperature and -5 "C. The cryogenic apparatus (Figure 1)was used for plate development at -77 "C. A viewing cabinet with long (366 nm) and short (254 nm) ultraviolet (UV) lamps (Brinkmann, Westbury, N.Y.) was used to locate the spots on the plate. A Varian XL-100 NMR Spectrometer with a Nicolet TT-100 Fourier transform unit was used for the NMR spectra. Streaks on the plate were made using a De Saga Autoliner (Brinkmann, Westbury, N.Y.). Reagents. All solvents used were glass-distilled (Burdick and Jackson, Muskegon, Mich.). Deuterated solvents were obtained from Merck & Co., Inc. (Rahway, N.J.). Nitrosamines were synthesized at the Frederick Cancer Research Center. Drummond micropipets were used for spotting the sample solutions on silica gel plates, EM silica gel 60F-254 (Brinkmann, Westbury, N.Y.). Procedure. Solutions of the nitrosamines ( 5 mg/mL) were made in ethyl acetate. Plates were spotted (streaked) at room temperature after which they were cooled to the development temperature before they were developed for approximately 12 cm in a saturated tank or cryogenic apparatus (Figure 1) which had been equilibrated at the required temperature. The solvent systems used were diethyl ether or hexane/tetrahydrofuran (3:l). After development, the plate was taken out and covered with a clear glass plate to shield the sample from UV light. A 1-cm channel at the edge of the plate was left uncovered. The plate was then viewed under short UV and the bands were marked with a pencil. While still wet, the bands were scraped off the plate, one at a time, as fast as possible to avoid plate warmup and transferred to a cold flask which was kept at -77 "C in an acetone-dry ice bath. The flask was capped to prevent acetone contamination. The separated conformers were extracted from the silica gel by adding cold methylene chloride and the flask was shaken for 5 min. After settling for a few minutes, the methylene chloride extract was transferred to test tubes which were kept at -77 "C. The test tubes were evacuated under reduced pressure (0.1-0.005mm Hg) until the solvents were removed. The residue was then dissolved in cold deuterated chloroform and an NMR spectrum was obtained at -20 "C or -40 "C. RESULTS AND DISCUSSION TLC of 1,4-dinitrosopiperazine (DNPZ) and its cis-2,6dimethyl-(2,6-DMDNPZ), truns-2,5-dimethyl-(2,5-DMDNPZ), and 2-methyl (2-MDNPZ) derivatives yielded one spot each a t room temperature and a t -5 "C when spotted on silica gel plates and developed in either diethyl ether or hexaneltetrahydrofuran (3:l). Published NMR data indicate the presence of two isomers for DNPZ (8)and 2,6-DMDNPZ (8), three isomers for 2,5-DMDNPZ (8), and four isomers for 2-MDNPZ (9). No separation of the isomers occurred even a t -20 "C, so the plates were then continuously developed a t -77 "C, obtained by using a dry ice-acetone bath in a cryogenic apparatus developed in our laboratory (Figure 1). T h e cryogenic apparatus is a closed unit to prevent water from the atmosphere from condensing on the plate, which would make evaporation of the solvent from the plate difficult, and thus interfere with the separation. Initially, the separation of 2,B-DMDNPZ and 2-MDNPZ a t -77 "C under continuous development in tetrahydrofuran-hexane (1:3) gave two spots for 2,6-DMDNPZ and three spots for 2-MDNPZ after 2 h. In another experiment, the four compounds 2-MDNPZ, 2,60 1979 American Chemical Society
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Figure 1. Coldlcontinuous development apparatus A 1
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Flgure 2. Comparison of the separation of nitrosamines: (1) DNPZ, (2) 2-MDNPZ, (3) P,B-DMDNPZ, (4) 2,6-DMDNPZ, (5) NMEPO, and (6) 3,5-DMN4P at (A) room temperature, (B) -5 OC, and (C) -77 OC
DMDNPZ, 2,5-DMDNPZ, and DNPZ were each spotted on three plates and the plates were developed once in diethyl ether a t room temperature, -5 "C, and -77 "C. Separation of the isomers was achieved only at -77 "C (Figure 2). Continuous development a t -77 "C for 2 h improved the resolution. I t is apparent, therefore, that continuous development at low temperatures significantly improved the separation of these compounds. In certain cases it may not be necessary to use low temperatures. For example, N-nitroso-3-methyl-4-piperidone (NMEPO) was separated into two spots (Rf X 100 = 38 and 50) when chromatographed on silica gel plates and developed in petroleum ether-ethyl acetate (3:2) a t -5 "C. Two experiments were carried out to establish that the two resolved spots are conformational isomers. First, a solution of NMEPO was spotted onto a cold (-5 "C) TLC plate and developed as described above to give two spots (lower temperatures were found to give a better separation). The plate was then left a t room temperature for 10 min after which it was cooled in the refrigerator, placed in the cold tank, and developed at 90' to the first development. After development, each spot was resolved into two spots which had the same relative R, values as initially found. This suggested that the individual conformers re-equilibrated at room temperature to the two conformational isomers. When the solution was spotted and developed in both directions at a cold temperature, only two spots were observed. Proton NMR spectra at -20 "C of the two isolated compounds, eluted off the plate a t low temperature (Figure 3), indicated that each spot contained a single isomer of NMEPO and not decomposition products. Relative intensities of the methyl doublets a t 6 = 1.14 and 1.22 (Figure 3) clearly show that each sample is BO-90% conformationally pure. Figure 3D indicates that the isomers re-equilibrate at room temperature with a half-life of 20 to
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Flgure 3. 100-MHr 'H NMR spectra of N-nitros& methyl4piperidone at -20 OC in CDCI,. (A) Equilibrium mixture of two isomers. (B) Low R,isomer isolated by cryogenic TLC. (C) High R,Isomer isolated by cryogenic TLC. (D) Same sample as C, after warming to 22 OC for 20 min. Impurity peaks at 6 = 1.8, 2.1, and 5.3 are water, acetone, and methylene chloride, respectively
30 min. Each of the samples whose spectra are shown in Figure 3B and 3C gave spectra indistinguishable from Figure 3A when allowed to equilibrate to room temperature for several hours. The spectrum in Figure 3C indicated that this is the anti-3-equatorial-methyl isomer in a fairly normal chair conformation. The resonances of the a protons can be separated into four distinct multiplets, centered a t 6 = 3.4, 3.9, 4.8, and 5.1. On the basis of the relative chemical shifts of a protons in other cyclic nitrosamines (IO),we assigned the multiplet at 6 = 4 to an axial proton, anti to the nitroso. This proton is clearly coupled to only two other protons, with coupling constants of 13.3 and 10.6 Hz,which are typical values for germinal and axial-axial coupling constants, respectively (11). This proton must, therefore, be adjacent to the proton bearing the methyl group, and the methyl must
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barriers to ring inversion as well as nitroso group rotation will be published separately. For the four piperazines, DNPZ, 2-MDNPZ, 2,5-DMDNPZ, and 2,6-DMDNPZ which were separated a t -77 "C, TLC indicated that the component conformers re-equilibrated at room temperature. When the plate was turned 90" and developed again, each spot separated into the same number of spots as obtained in the first development. Figure 4 shows the proton NMR spectra of the two isomers of 2,B-DMDNPZ along with a spectrum of the equilibrium mixture. The four methyl doublets in the region 6 = 1.0-1.6 show that good separation of the isomers was achieved a t -77 OC, although some diethyl ether, used as developing solvent, remained in one of the samples (Figure 4B). The chemical shifts of the methyl signals indicated that the isomer with high R, (Figure 4B) has the two nitroso groups cis to each other (IO),while the bottom spot (Figure 4C) has the two nitroso groups trans to each other (8). When the separated isomers were warmed up to -16 "C for 20 min, the proton NMR spectrum showed that the isomers had nearly equilibrated. It is apparent from the above results that cryogenic TLC is a practical way to separate cyclic nitroso conformational isomers. Better resolution is achieved when cryogenic/ continuous development is used. Combination of cryogenic TLC and NMR spectroscopy would make possible kinetic studies of nitroso group rotations in cyclic nitrosamines.
LITERATURE CITED
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D. C. Abbott, H. Egan, and J. Thornson, J. Chromatcgr., 16, 481 (1964). E. Stahl, Angew. Chem. Int. Ed. fngl., 3, 784 (1964). E. Stahl, Arch. fharmaz., (Weinheim, G e r . ) , 297, 500 (1964). R. F. Henderson and M. H. Clayton, Anal. Biochem., 70 440 (1976). (5) B. Liberek, J. Augustyniak, J. Cirkowski, K . Plucinska, and K. Stachowlak, J . Chromatogr.: 95, 223 (1974). (6) W. T. Iwaoka, T. Hansen, S. T. Hsieh, and M. C. Archer, J. Chromatogr.. 103. 349 11975). (7) A:knschrak, H. Munch, and A. Mattus, Angew. Chem., 78, 751 (1966). (8) D. Hofner, D. S.Stephenson, and G. Birnsch, J . Magn. Reson., 32, 131 (1978). (9) G. Singer, unpublished results. (10) Y. L. Chow and C. J. Colon, Can.,!. Chem., 46, 2827 (1968). (11) L. M. Jackson, and S. Sternhell, Applications of Nuclear Magnetic Resonance Spectroscopy to Organic Chemistry ', Pergamon Press, Elmsford, N.Y., 1969. (1) (2) (3) (4)
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Figure 4. 100-MHz 'H NMR spectra of dinitrose2,6dimethylpiperazine. (A) Equilibrium mixture at 25" C. (B) High R,isomer at -40 "C,isolated by cryogenic TLC. Impurity peaks are acetone at 6 = 2.1, water 6 = 1.8, and diethyl ether (developing solvent) at 6 = 3.4 and 1.2. (C) Low R, isomer at -40 "C, isolated by cryogenic TLC. Impurity peaks are acetone at 6 = 2.1 and water at 6 = 1.8
be equatorial. The spectrum, Figure 3B, is not so readily analyzed, but we believe that the nitroso group is anti to the nitroso with the ring in a flexible twist conformation. A full discussion of the spectra and factors causing the increased
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RECEIVED for review July 9,1979. accepted August 17,1979. Presented in part at the 30th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, 1979. This work was supported by Contract N01CO-75380 with the National Cancer Institute, NIH, Bethesda, Md. 20205.
