フルオレセインクロライド-塩化亜鉛-アミン系の加熱溶融反応と薄層クロマトグラフィーによる脂肪族窒素化合物の超微量分析

The method is based on the production of a certain rhodamine dye by the thermal reaction in fluorescein chloride-zinc chloride-amine system followed by the densitometry of thin-layer chromatogram of the product.<BR>In order to determine the ultramicro amounts of aliphatic nitrogen compounds, the various conditions such as temperature(Fig. 4), time(Fig. 5), amount of zinc chloride(Fig. 6) and of fluorescein chloride(Fig. 7) in the thermal reaction were investigated. The effect of hydrochloric acid concentration(Fig. 8), temperature (Fig. 9) and time(Fig. 10) in treating the product of the thermal reaction were also investigated.<BR>The separation and determination of the product were made by silica gel thin-layer chromatography and densitometry utilizing a dual wavelength spectrophotometer.<BR>The recommended procedure is as follows: To a test tube(Fig. 1) 10 μl of methanol or chloroform solution containing 1 to 10 μg of sample, 40 μl of chloroform solution containing 1.5% fluorescein chloride and 20 μl of methanol solution containing 3.0% zinc chloride are added. The solution is evaporated to dryness, and the residue is then heated for 5 min at 260°C.<BR>After being cooled the residue is dissolved in 50 μl of methanol solution containing 1.5% hydrochloric acid. The solution is heated for few minutes at 80°C. The solution is cooled to room temperature, and 120 μl of it is spotted onto the silica gel thin-layer, which is developed with n-buthanol-acetic acid-chloroform-water(10 : 1.5 : 1.5 : 1.5).<BR>As shown in Fig. 2 the thin-layer chromatogram (densitogram) determined by a dual wavelength spectrophotometer, which is superior to a single wavelength spectrophotometer in linearity of base line, and R_f values and peak areas are calculated. If the sample contains ammonia or an amine, the densitogram in dicates the peak of the rhodamine dye produced in the thermal reaction. <BR>The carbon number of amines can be determined from the R_f value(Fig. 11 and Table II), and it is possible to determine ultramicro amounts of aliphatic amines by the calibration curve(the amount of amine plotted against the peak area of densitogram). The calibration curve showed good linearity in the range of 0 to 0.1 μg as nitrogen, and the sensitivity(peak area divided by mass of nitrogen, that is, proportionality constant in calibration line) calculated from the calibration curve was 23× 10⁴ mm²/μg.<BR>Furthermore, the method is applicable to aliphatic nitriles, acid amides, nitro compounds and hydrazines, if the compounds can be convertedto amines or ammonia by the pretreatment such as hydrolysis or redox reaction (Table II).