Extinction coefficient, optical principles

Fairly wide use has been made of preparative gel electrophoresis in protein chemistry, and in principle there is no reason why the same procedures should not be adopted for use with nucleic acids which have the advantage that much may be accomplished with very small quantities of purified material. Thus, it is relatively easy in many situations to introduce radioactive label at very high levels and specific activity, and the use of for this purpose offers a degree of sensitivity that cannot be matched in work on proteins. The extinction coefficients of nucleic acids are also very high in the ultraviolet, so that with say 20 pg in 1 ml or less it is possible to measure optical properties, thermal melting profiles, sedimentation coefficients, and even molecular weights by sedimentation equilibrium in an instrument equipped with scanner optics. Consequently, the sacrifice of resolution that, by a malign law of nature, always accompanies any attempt to scale up an analytical fractionation method is often at least partly avoided. 

In principle, a definitive identification of the optically detected species P430 and the species responsible for the EPR, iron-sulfur signal would be best made with spectro-kinetic measurements. However, this approach is limited by the fact that although spectro-kinetic measurements may be performed on the optical species P430 with relatively high time resolution, in spite of its rather small extinction coefficient. 

Assuming an exact knowledge of the nature of the electronic transition or vibration associated with each absorption band, and assuming that the effect of the electrostatic field of a surface on these transitions can be calculated reliably, it should be possible, in principle at least, to deduce the orientation of the molecule on the surface from observation of the change in extinction coefficient with coverage. Unfortunately, our knowledge and understanding of optical transition and of the effects of an electrostatic field on these transitions is not sufficiently developed to permit such detailed conclusions. 

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