Is direct spectrophotometric determination of chlorophyll in pigment extracts of tissues under different physiological conditions valid?

Direct spectrophotometric determination of chlorophyll content from methanol extracts of young and senescent leaves of $\text{Thunbergia}\text{grandiflora}$ showed 29 and 16%, respectively, of mature leaves. Spectrophotometric estimation in the same extracts after separation of chlorophyll by thin layer chromatography or by direct fluorometric determination showed only 15 and 0.5% of mature leaves, indicating an error of 4 to 40 fold. In the above wide range of absorbance (0.5 to 100%) the lack of linearity between absorption and concentration resulted in additional error. Bound chlorophyll remaining in the pellet, not extractable by any solvent system, was also reported to vary depending on the physiological conditions of the tissue (Plant Physiol. 51, 660–666). Therefore, it is pointed out that the aforementioned errors observed in direct spectrophotometric determinations could be checked by monitoring simultaneously the cell or plastid suspensions $\text{in}\text{vivo}$ by fluorometry without any extraction.