Abstract
Some of the basic problems in the quantitative analysis of pregnanediol, using pregnanediol obtained from pregnancy urine accoding to our extraction and isotation procedures, were carried out.
1. H2SO4 chromogen of pregnanediol : The absorption curves of isolated substances obtained from pregnancy urine (5 liters of each), which had been dissolved in 5.0 ml. of 95% H2SO4 and left at room temperature for 30 minutes, were compared. All of the spectrums of the specimens exhibited peaks at 300 mμ. and 420 mμ. and the bottom at 390 mμ.. We, therefore, found out that all of our extracted substances were identical.
2. Relationship between concentration of H2SO4 and optical density of H2SO4 chromogen of pregnanediol : The optical density of H2SO4 chromogen of pregnanediol was elevated as the concentration of H2SO4 was increased in both the following conditions ; when 32.4 μg. of the material was left at room temperature for 20 minutes in 5.0 ml. of 65%, 95% and 95% H2SO4 and when 21.6 μg. was incubated at 100°C for 20 minutes in 5.0 ml. of each of the above percentages of H2SO4.
3. Relationship between duration of incubation and optical density of H2SO4 chromogen of pregnanediol : 32.4 μg. of the material was incubated at 100°C in 5.0 ml. of each of the above mentioned percentages of H2SO4. The optical density was elevated as the concentration increased for incubation of less than 20 minutes duration, but the optical density became constant as the duration was prolonged for more than 20 minutes.
4. Relationship between lapse after colour development and optical density : The optical density of H2SO4 chromogen, when 27 μg. of the material in 5.0 ml. of 95% H2SO4 was not incubated, gradually increased after colour development, and became constant after 30 minutes for 30 minutes. When the material was incubated for 20 minutes at 100°C and then was cooled, the optical density became constant just after cooling for 60 minutes.
5. Relationship between duration of incubation and amount of material : When different amounts from 10.8 μg. to 43.2 μg of the material were incubated at 100°C in 5.0 ml. of 95% H2SO2, the optical density of each material remained constant after incubation for 20 minutes. The absorption curves of 27 μg. of the material incubated for 5, 10 and 20 minutes were different from that of substances not incubated in the region under 390 mμ., but were not markedly different in the region above 390 mμ.. According to the calibration curve between the above amounts of material and their H2SO4 chromogen, we found that pregnanediol was quantitatively estimated in much smaller amounts after incubation for 20 minutes at 100°C than after no incubation.
