1. A trial by means of paper chromatography on the studies in regard to the variation and formation of leaf pigments was made, using three strains or lines of rice (Zuiho-green leaf, Murasaki-ine-purple leaf and Kogane-nishiki-yellow leaf).
2. The extraction of pigments was carried out with a solution of methanol, acetone, benzene in 4:1:1 ratios. Approximately 24 hours were elapsed and after filtration the solvent was evaporated under the low pressure, and thus the residual crude pigment was obtained. This was solved again with ether, and thus used as material. The filter paper used was Toyo Roshi No. 50 (1×40cm) and the material spotted at the situation of 6cm from the surface of solution. Developmental solution was of toluol and petroleum benzine (2:1), and by the ascending method the experiments were carried out 40 minutes at a temperature of 4°C.
3. The pigments were separated clearly in 5 spot areas (Fig. 1) in order of chlorophyll b (yellow green), chlophyll a (blue green), 2 sorts of Xanthophyll (yellow) and carotene (orange yellow), and thus Rf values and coloured areas were measured. Although the Rf value of carotene was almost constant, other pigments increased rectilinearly according to their sample amounts (Tab. 1, Fig. 2).
4. Owing to the test of observed error in coloured area a variance analysis was done for the measured values, denoting very small in the observed error in the same cylinder as shown in Table 2. The relation between sample amount and coloured area for respective pigments (Tab. 3, Figs. 37) differs in the situation and slope of curves according to their materials, but the curves which select the most adequate experimental formula seem to be almost similar. Thus three strains will be discussed together in treatment. Consequently the related formula thus obtained will be applicable to those other then these three strains.
5, The authors subjected the data to x
2-test (Tab. 4) whether the follow-ing formulae are most applicable to the relation between sample amount (x) and coloured area (y) or not.
1) y=K
1x+K
22) y=K
1logx+K
23) log y=K
1logx+K
2where K
1 and K
2 are constants. The result shows that (2) formula is most adapted for carotene and xanthophyll, and (3) formula for chlorophyll, (1) formula being conformable also to chlorophyll b.
6. For the sake of the analysis of relative contents, Zuiho with green leaves under the usual culture was used as control, and the constants of respective pigments were calculated by the most applicable experimental formula for this material. Thus the nomogram (Fig. 8) was plotted, showing relation between pigment amount and coloured area. The actual values the from other strains will be put in this nomogram and provisionally the calculated γ of crude pigments as those of the control Zuiho will be deter-mined (Tab. 5).
7. To avoid the error in the case of actual measurement, the pigment amounts will be placed in the range of 40100 calculated γ, adjusting the dilution of sample density. And simply and speedily the experiments will be carried out by the extraction with methanol, acetone and benzene, whereas the upper clear portion of extraction standing for a while after the mixing is soon used as sample solution. Moreover, in this method the test will be safely carried out even with the sample amount of 0.5gr. fresh leaves.
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