(1) Quantitative determination of vitamin D by alumina or silicagel thin-layer chromatography (TLC) using benzene・acetone・chloroform (10 : 1 : 1) as a developing solvent system is possible in the range of 5 to 50μg and the necessary conditions were described. (2) Absorption spectra in ultraviolet region of vitamin D and related compounds and those of the colored solutions by Nield reagent were determined. No correlations were found between the two. (3) Those showing the absorption spectra of the Nield reaction similar to that of vitamin D were isoergocalciferol, isotachysterol_2,tachysterol_2 and 5,6-trans ergocalciferol. (4) In the chromatogram of TLC those having the Rf values near that of vitamin D were tachysterol_2,isotachysterol_2,5,6-trans ergocalciferol, isoergocalciferol, retinol, ergosterol, 7-dehydrocholesterol, cholesterol, and lumisterol. Of these compounds, cholesterol, ergosterol and 7-dehydrocholesterol were completely removed by digittonin treatment. (5) Of these compounds, the one having the most effect on the determination of vitamin D is tachysterol. It seems to be somewhat decomposed by digitonin treatment and TLC but it will be difficult to be fractionated from vitamin D in the presence of much tachysterol.
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