注記 |
Methods of colorimetric estimation of in organic nitrogenous compounds and some oximes so far used in our laboratory were discussed and some supplementary experiments were carried out. At present the methods described seemed to be the best. Standard curves and calibrating equations were also determined by means of a Hitachi electric colorimeter, type EPO-A , with filter and cells of 10 mm light pass or a Hitachi spectrophotometer , type EPU-2, with cells of 10 mm light pass. Outlines are as follows. Nitrate Five ml of sample solution were mixed with 15 ml of 85 % H_2SO_4 and cooled . Then 1 ml of 1 % 2, 4-xylenol (in glacial acetic acid) was added. After 30 min. at room temperature, the mixture was transferred into a separate funnel of 200 ml with aid of H_2O making 100 to 150 ml of final volumes. Ten ml of toluene were added, shaken vigorously and H_2O layer was discarded . Into remaining toluene, 50 ml of H_2O were added, shaken and removed . Washing was repeated once more. Finally 5 ml of 0.4 N NaOH were added to toluene and mixed gently. NaOH layer become yellow was separated from toluene. Intensity of color of alkaline solution was estimated with a spectrophotometer at 410 mμ. Relation between extinction and concentration of NO_3 was shown in Fig. 1. From this Fig. 1, the equation (a) was obtained to calibrate the amounts of NO_3-N in sample. NO_3-N r=21.3 E --2.13.........(a) Since NO_2 also develops yellow color, it should be removed beforehand if sample is contaminated by NO_2. Prior to coloration, 5 ml of sample solution were mixed with 1 ml of glacial acetic acid and 1 ml of 10 % urea solution and the mixture was heated at 100℃ for 10 min. Nitrite Routine Griess' reagent was employed. Into 10 ml of NO2 solution, 1 ml of 1 % sulfanilic acid (in 25 % acetic acid) and 1 ml of 0.4 % a-naphthylamine (in dil. HC1) were added. After 5 to 20 min. at room temperature, red color developed was estimated with electric colorimeter using green filter or with spectrophotometer at 525 m/a. Extinction is proportional to the concentration of NO_2 as shown in Fig. 2. Calibrating equations (b) and (c) were obtained from Fig. 2. NO_2-N r=4.9 E-0.05.........(b) (electric colorimeter) NO_2-N r=4.2 E ...............(c) (spectrophotometer) Hyponitrite One ml of 1 % Na-sulfanilate, 0.3 ml of 1.3 % iodine solution (in 2 % KI) and 0.15 ml of 0.5 N NaOH were added into 10 ml of hyponitrite solution. After standing at room temperature for 30 min., 1 ml of 30 % acetic acid, 0.15 ml of 0.5 N HCl, 0.3 ml of 0.1 N Na_2S_2O_3 and 1 ml of a-naphthylamine were added. As in the case of NO_2, color intensity was determined. Extinction was shown in Fig. 3 and calibrating equations (d) and (e). N_2O_2-N r=21.0 E-0.06.........(d) (electric colorimeter) N_2O_2-N r=12.12 E ...............(e) (spectrophoto meter) Red color develops with NO_2, NH_2OH and some oximes by this procedure. When sample contains all or one of substances cited above too, another aliquot of sample was heated at 100℃ for 5 min. to destroy N_2O_2 before coloration. Difference between color intensities by both samples with and without heating corresponds to N_2O_2. Hydroxylamine Two kinds of methods were used with Griess' reagent or 8-hydroxyquinoline. Procedure with former was connected with iodine oxidation in acidic, alkaline or neutral media. (I) Acidic iodine oxidation method. Ten ml of sample were mixed with 1 ml o f 1 % sulfanilic acid and 0.3 ml of 1.3 % iodine solution (in glacial acetic acid). The mixture was heated by immersing it into hot water at 90℃ for 45 sec. and rapidly cooled with running tap water. To remove excess iodine, 0.3 ml of 0.1 N Na_2S_2O_3 was added. Finally 1 ml of a-naphthylamine was added to develop red color. Fig. 4 shows the direct relation between extinction and concentration of NH_2OH and equations (f) and (g) give the amounts NH_2OH-N in sample solution. NH_2OH-N r=6.2 E-0.05.........(f) (electric colorimeter) NH_2OH-N r=4.75 E ............(g) (spectrophotometer) (II) Alkaline iodine oxidation method. The procedure is same as that for N_2O_2 estimation described above. Fig. 5 and equations (h) and (i) were obtained. NH_2OH-N r=8.0E .........(h) (electric colorimeter) NH_2OH-N r=5.6 E......... (i) (spectrophotometer ) (III) Neutral iodine oxidation method. This method was devised to assay NH_2OH contaminated with some oximes, since both substances develop red color by means of the acidic or alkaline iodine oxidation method cited above. To a mixture of 1 ml of 1.5 M Na-acetate, 1 ml of 1 % Na-sulfanilate and 0.3 ml of 1.3 % iodine (in KI) solution, 5 ml of NH_2OH solution were added. After shaking quickly, 0.3 ml of Na_2S_2O_3 were added at once to remove excess iodine. Then 3 ml of 0.5 N HCI, 1 ml of 30 % acetic acid and 1 ml of a-naphthylamine were added. Fig. 6 is relation between extinction and concentration of NH_2OH and (j) and (k) are calibrating equations. NH_2OH-N r=12.5 E-0.125.........( j ) (electric colorimeter) NH_2OH-N r=10 E ..................(k) (spectrophotometer) (IV) 8-Hydroxyqiunoline method. Since NO_2, N_2O_2 and oximes do not develop the color deffering from the procedures with Griess' reagent, this method is most suitable for estimation of NH_2OH. Two ml of sample solution were mixed with each 1 ml of 0.2 M Tris buffer of pH 7.5, 1 % 8- hydroxyquinoline (in alcohol) and 2 N Na2CO3 solution. Green color developed was determined with spectrophotometer at 700 mg, after standing at room temperature over 15℃ for 60 min . Result is shown in Fig. 7 or equation (1). NH_2OH-N r-3.0 E-0.02.........(1) (spectrophotometer) Oximes Oximes can be oxidized into NO_2 with iodine directly or indirectly connected with acid hydrolysis. (I) Direct oxidation method. Acetoxime was estimated by acid iodine oxidation method of NH_2OH. Alkaline iodine oxidation method of NH2OH also can be applied to the estimation of some oximes such as pyruvic acid-, cr-ketoglutaric acid-, oxalacetic acid-, acetaldehyde- and benzaldehyde- oxime, but not to acetoxime or sugar oximes. Standard curves and calibrating equations of NH_2OH with iodine oxidation methods can be used for the assay of oximes by the same procedures. (II) Acid hydrolysis method. This method is mostly employed in order to estimate sugar oximes. A mixture of 10 ml of oxime solution and 0.2 ml of 1 N H_2SO_4 were heated at 100℃ for 5 min. to hydrolyse oximes. Then NH_2OH formed was estimated by means of acidic iodine oxidation method. Since rates of color development vary with kinds of oximes, standard curves or calibrating equation should be determined on each oxime. A example for glucose oxime is shown in Fig. 8 or equation (m). Oxime - Nr=5.9 E.........(m) (electric colorimeter) Ammonia Coloration of ammonia with indophenol method is more sensitive than that with Nessler's reagent. Into 1 ml of sample solution, 1 ml of 5 06 alkaline phenol (in 2 % NaOH), 7 ml of 0.1 M Na2HPO4 (pH 9.8 adjusted with 1 N NaOH), 0.5 ml of 0.05 % Na-nitroprusside and 0.5 ml of antiformin (4 % effective Cl) solution were added. After 30 min. at or over 20℃, blue color developed was determined by electric colorimeter with red filter. Since routine laboratory distilled water also develops pale blue color by this procedure, color intensity must be corrected. Furthermore, color intensities are fluctuated with some other factors, it is more suitable to make a standard curve with (NH4)2SO4 of known concentrations on the same time. Blue color develops with NH2OH too. However, the color intensity was reduced to negligible without effecting on the coloration of ammonia, when buffer was first mixed with sample prior to addition of phenol. Of course, no color develops with NHOH after treatment of sample in a Conway's unit.
|
|