LD Spectroscopy

Linear dichroism is defined as the differential absorption of linearly polarized light

AI I is the absorbance of the sample when the light is polarized parallel to a reference axis, and A? is the absorbance of light which is polarized perpendicular to this axis. The strength of the absorption depends on the orientation of the electric field vector of the light and the transition moment of the chromophore - parallel orientation results in maximum absorption whereas perpendicular orientation leads to zero absorption. By dividing the LD value by the absorbance of the un-oriented sample under isotropic conditions (Aiso), the ''reduced'' linear dichroism (LDr), i.e. the wavelength-dependent LD, is obtained (Eq. 7) [36].

The LDr correlates with the orientation of the transition moment of the dye relative to the reference axis, as quantified by the angle a. LDr is also proportional to an orientation factor S (S = 1 denotes perfect alignment of the dye, S = 0 random orientation). For an isolated, non-overlapping transition, Eq. (7) establishes the correlation between LDr, a and S. These definitions lead to the qualitative rule that with an angle a > 55°, a negative LD signal is observed, whereas with a < 55°, a positive signal appears in the spectrum. Thus, with an appropriate set-up the orientation of a chromophore relative to a reference axis can be determined.

From Eq. (7) it is obvious that in an isotropic medium a LD signal cannot be detected because of the statistical orientation of the transition moments under these conditions. Thus, methods are required to promote preferential orientation of molecules. For LD experiments two general approaches have been established:

light polarized perpendicular relative to the reference axis light polarized perpendicular relative to the reference axis

DNA aligned along the hydrodynamic field

light polarized parallel relative to the reference axis

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