Discussion 



Dr. Mason: One of the major purposes of Dr. Spruit's work has evi- 

 dently been to characterize Cijpridina luciferin by means of system- 

 atic study of the absorption spectra of certain chromophores. Since 

 very small quantities of substance are required for the measurement 

 of absorption spectra, the method has obviously a wide application 

 to the determination of structure of natural products, and it is of 

 interest to consider what its limitations may be. 



Aside from technical problems, such as the establishment of homo- 

 geneity, the principal limitation lies in the presupposition that it is 

 possible to assign a structure to a light-absorbing molecule with a 

 high degree of confidence when only the absorption spectrum is avail- 

 able. In so far as the ultraviolet and \ isible regions of the spectrum 

 are concerned, very large classes of compounds tend to absorb light 

 in certain well-defined regions such as 260 to 280 millimicrons. If the 

 substance we wash to characterize displays a single absorption band 

 in this region, the confidence with which we can assign a structure 

 to it is very low indeed, even if the correspondence between the 

 absorption spectra of the known and unknown substances is high. 

 If the unknown substance displays two absorption maxima in the 

 ultraviolet and visible region and the correspondence between the 

 absorption spectra of the known and unknown substances is again 

 relatively exact, the degree of confidence in the structural identifica- 

 tion rises correspondingly. Extending this hue of thought, the larger 

 the number of corresponding uniquenesses between the spectra of 

 known and unknown substances, the higher the degree of confidence 

 we may have in the identification. Characteristic absorption maxima 

 for a single compound can be multiplied by observing spectral changes 

 which can be induced by changes in environment or by frank reac- 

 tion, that is, alterations in pH, in solvent type, or by autoxidation. 



There is one important reservation which should be made in regard 

 to the amount of structural detail which is revealed by absorption 

 spectra in the ultra\dolet and visible regions. In order to be spectro- 

 scopically detectable, each atom or system of atoms in an absorbing 

 molecule must have the capacit\' to affect, detectably and character- 

 istically, the levels of excitation to which the molecular orbitals can 

 be raised. Inasmuch as there are very large numbers of possible alter- 



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