Separation of Aromatic Nitro Compounds by One-dimensional Dual Band Thin-Layer Chromatography and High Speed Liquid Chromatography

Abstract

To develop a microanalytical method for aromatic nitro compounds, separation behavior of these compounds have been studied by a dual band thin-layer chromatography (TLC) and a high speed liquid chromatography (HPLC). In the former chromatography, aromatic nitro compounds were applied to the kieselguhr layer of the dual band TLC plate which was composed of kieselguhr layer (4×20 cm) and 26 % acetylated cellulose layer (11×20 cm), and then developed to the 10 cm mark of the acetylated cellulose layer with methanol-ether-water (4: 4: 1, v/v). In the latter chromatography, separation behavior of aromatic nitro compounds were surveyed by the 2 separation system, Nucleosil 7C18 column (4.6×35 mm+4.6×250 mm)-acetonitrile-water (5: 5-9: 1, v/v) system and Nucleosil 5NO2 column (4.6×35 mm+4.6×250 mm)-n-hexane-benzene (5: 5-9: 1, v/v) system.
Separation pattern of aromatic nitro compounds in this TLC was very good as compared with that in an ordinary TLC, and several carcinogens tested such as 2-Nitrofluorene, 2, 5-Dinitrofluorene and 2, 7-Dinitrofluorene were easily separated each other. It was also found that a minor component in a sample could gather together easily from several TLC plates in order to analyze the component, because of high reproducibility of relative Rf values of aromatic nitro compounds in this TLC (Rf_{6-Nitroquinoline}=100).
In HPLC, retention time of a aromatic nitro compound decreased with the rise of column temperature. There was a linear relation between the logarithm of the retention time and the reciprocal of absolute temperature of column. Retention time of a compound decreased also logarithmically with the increment of acetonitrile content of the acetonitrile-water mobil phase or the increment of benzene content of the n-hexane-benzene mobil phase.
Separation pattern of aromatic nitro compounds was different in the both HPLC systems tested. The pattern was mainly governed by the number of benzene rings of the compounds in the Nucleosil 7C₁₈ column system. On the other hand, the pattern was mainly governed by the number of nitrogroups of the compounds in the Nucleosil 5NO₂ column system. Most of 35 aromatic nitro compounds tested could easily separate each other by the combination use of the 2 HPLC systems. It was also found that the combination use of TLC and HPLC was useful for analyzing some aromatic nitro compounds in the environment.